Martin Jc

Chatel Jm

Sémont A Effet

Alexandra Sémont
email: alexandra.semont@irsn.fr

A Lapiere

A L Orcid

Gabriel Fernandes

Jean-Marc Chatel
email: jean-marc.chatel@inrae.fr

STATE OF THE ART I. CANCERS AND RADIOTHERAPY 1. PELVIC CANCERS 2. THE ROLE OF RADIOTHERAPY IN THE THERAPEUTIC STRATEGY FOR PELVIC CANCERS

Keywords: Radiotherapy, Temporal dynamic study, Digestive toxicity, Colonic ecosystem, Colonic epithelial barrier, Microbiota, Shotgun metagenomic, Metabolome, in silico approaches, mathematical and statistical tools Faecalibacterium prausnitzii, prophylactic treatment, pelvic radiation disease, colonic epithelial barrier, inflammation, tuft cells

A. Principle of radiotherapy B. Application of radiotherapy in the context of pelvic cancers a. Radiotherapy objectives b. Type of radiotherapy and energy available for radiotherapy c

Thanks

First, I thank the jury members for agreeing to assess this thesis. I would like to thank Prof.

Céline Bourgier for agreeing to chair this thesis, Dr Anne-Judith Waligora and Prof. Xavier Treton for agreeing to act as reviewers, and Dr Martin Larsen and Dr Joël Doré for taking on the role of examiners.

I would also like to thank Dr Fabien Milliat and Dr Jean-Marc Chatel for supervising the thesis and Dr Alexandra Sémont for her guidance.

I would particularly like to thank you, Alexandra, for your guidance over the years. Your involvement in all parts of the project was a great help, and your invaluable advice was very useful in helping me to acquire rigour and a scientific methodology. I'd also like to thank you for all the good times we've shared at work and outside. Working alongside you was a real pleasure, and it contributed enormously to keeping very good memories of my thesis years. I would also like to thank Fabien for monitoring the project over the three years of his thesis.

Thank you for your availability to answer the various questions we may have had during this thesis. But also, thank you for sharing your opinion on the experiments, which enabled us to answer the scientific questions we wanted to answer, and for your help in analysing the results, which made it easier for us to interpret them. Your participation was a great help in moving the project forward.

Jean-Marc, thank you for your advice and your comments about the project, the experiments and the interpretation of the results, despite the difficulty that distance can bring. Mathieu, thank you for introducing me to the world of microbiota sequencing and for all the explanations you gave me, which enabled me to tackle the subject and better understand our results. Oscar, thank you for your invaluable help with the microbiota analyses. I'm well aware of the time you've taken on top of your thesis and I'm all the more grateful for that. Amine, I'd like to thank you for your expertise and availability in helping analyse data on the microbiota, the metabolome and the whole aspect of the association between the microbiota and the host.

Maâmar, Stéphane, thank you for your guidance on analysing the metabolome results and your advice on interpreting the results. I would like to express my thanks to everyone at the LRMed laboratory. It has been a pleasure to share all these years in your company. Noëlle, Christelle, Agnès, Olivier, Vincent, Alain, Christine, Claire, George and Valérie. Thank you for all your presentations on various subjects and your advice, expertise and comments. These have helped broaden my knowledge of radiobiology but also moved us forward in our project and made us think about things we hadn't thought of. A special mention to Agnès, Noëlle, Christelle and Claire, who helped us with all the questions relating to exposure to ionising radiation in the digestive tract. Christelle, for all the days off you, took to help us with our digestion protocols. Valérie, for your help during the last experiments of the thesis.Thank you Véronique for your help with all the administrative procedures and Chloé for always ensuring we had our equipment when needed.

Finally, I would like to express my deep gratitude to all the students in the department and those who have helped make my years at IRSN such a joy.

Of course, thank you to Martin, especially for sharing this difficult year (especially for you) and for your support in the face of the many announcements of new defeats. I'm convinced that next year will go better, especially as the season will start in parallel with the arrival of a new person in this world (future recruit?) who I hope will make better team choices than you... More sincerely, thank you for all your support during these years it was extremely pleasant to share it with you I am delighted to have met you. Lydia, thank you for raising the office level before the arrival of Batoul, who also made a huge contribution. I thank you both for our rich conversations and your support and encouragement. I wish you both all the best for the future.

Lydia, congratulations once again on your acceptance to medical school. You will be a terrific doctor, without a doubt. Thank you to Calixte for participating in the project, and congratulations on your thesis. I do not doubt that you will succeed, especially with Alexandra's guidance and the help of Valérie and Fabien. You will be well looked after. Thank to Lucie for the good times, although the time we shared was short. Claire, Anne-Laure and Sarmini I'd like to thank you for your good mood and your contribution to all the latest Adin events, and Nathalie, I'd like to say a special thank you for your very pleasant presence and for your contribution to the project during your intership. It was real pleasure to work alongside you. Sarah, starting together with Lydia has been a wonderful experience, and it was a great pleasure to share it with both of you. I want to extend a special thank you for your dedication to improving student conditions within IRSN, fostering a positive atmosphere in the labs, and organizing various events to bring people together. Manon, I would like to thank you for your unwavering positivity and the abundant joy you bring to the entire team. Your laughter is always a pleasure to hear and it contributes greatly to the pleasant atmosphere within the service. Thank you, Lucie, I appreciate your discreet yet insightful presence and intesting conversations you bring. Goran, thanks for making the task easier, so thank to exist. Of course, Quentin, I am grateful for our precious moments. Meeting you last year was one of the best thing of the year. Please do not judge my English. I hope to keep our almost daily photo routine.

Thank Yoann for all the first irradiations and your company over these years, it is always a pleasure to share conversations with you and always instructive and thank you to Miray for taking over. You both participate in making the irradiation session pleasant.

Alexia, I want to express my heartfelt gratitude for passing on the torch of thesis to me in the best possible way. Thank you for introducing me to various techniques and your delightful presence throughout the years since we first met. You have been a true source of support for me. Thanks to Ségolène and Clément for your presence during your end of PhD.

For all my precious friends, thank you so much for your support all these years knowing you.

You are all extraordinary people and I hope so much you are conscient of that.

Of course, Anna and Elsa, since birth, every moment shared with you has been precious and unique. Elsa, you know what I would say: you mean everything to me. Thank you, Louisa, for all our conversations, which have been instructive and fueled my curiosity about various subjects. All the memories we've shared together are happy moments. Nicolas, since all this time, thank you for sharing your passion and helping to learn many things, especially about art.

Hanna thank you for your craziness and generosity; being in your presence always means great laughter. Emilie, thank you so much for initiating me to all internet culture; this helps me a lot in communication with certain people, and accessories amuse me a lot. Adam, the only image of you makes me smile, darling, thank for your consistently well-delivered humour. Takumi, thank you for all the moments we've shared, wherever and whenever, always filled with laughter and joyful thoughts. Roman, thank you for your commitment to all the enriching conversations. Same for Xavier, despite the distance, I hope to see you soon. To Zoé, thank you for all our shared moments that are incredibly precious and always very enlightening. And for all the goofing around we've done, it's a real pleasure and a great privilege to have you as one of my closest friends. This year marks the end of our studies together, even though they are in very different fields, it's another common thing we share. Sandrine, though far away, you are never far from my thoughts. Each year, new memories are created, bringing me immense happiness. Thank you for being there for such a long time. Charkit, thank you for being there all these years, for your good humor, ability to always have fun, and dedication and motivation.

It's been a privilege to see each other grow. All three of you are a great source of inspiration.

Pierre, thank you for all the help you have provided over the years, for the connections you have created, which have allowed me to meet people who are dear to me today. Thank you for your immense generosity, and I hope to see you soon with Eleonore and Jean. Marie, thank you for all the years we have shared by your side. You have been a true support and driving force during our studies. Laughter is the first thing that comes to mind when I think of you, so thank you for all those endless moments of laughter and your positivity. Antoine, thank you for your curiosity about a wide range of subjects, which allows us to learn something new with each interaction. Also, thank you for your humor and for sharing your experiences in various domains you have explored. And of course, Nico, thank you for making us all laugh with your incredible stories that we will never forget. Thank you also for being a great companion for all sorts of activities, except for Times Up, and for sharing the burden of roneo tasks. Adrien, thank you for all the insights you have provided regarding French history and other subjects.

Your presence is a great comfort, and it's always a real pleasure, despite our disagreements that will probably never be resolved because I love to annoy you with it. Romain, thank you for all our interesting and sometimes quirky conversations.

Yoann, you are the most pleasant company, and I thank you for all your advice. It's been a great privilege to evolve by your side. Thank you for your unwavering support during these years of the thesis, your kindness, availability, and attentive listening. And also, thank you for all the moments that allow me to relax, explore other disciplines, and laugh every day.

To my family, Mom, Dad, Arturo, Loanne, Gilles, Hélène, you are my greatest source of inspiration. I cherish every moment spent by your side and fully enjoy it. I won't elaborate further, but I hope you know you are all wonderful individuals. I am extremely grateful to have grown up in the rich and extraordinary ecosystem you created. You have all my love and gratitude.

ANNEXE 220

Table of abbreviations

Preface

Radiotherapy is an essential therapy in cancer treatment and is used in many cancer protocols for different types of cancer. Radiotherapy can serve different purposes and thus occur at other times in the cancer protocol. As it is a widely used treatment, knowing its advantages and limitations is essential. The benefit of radiotherapy is its effectiveness in killing cancer cells.

The disadvantage is that it does not differentiate between cancerous and healthy cells, as ionising radiation puts its energy on both types of cells, with the result that radiotherapy helps to kill healthy cells, causing damage to the organs around the tumour. As with any treatment, a benefit/risk ratio is considered before prescribing radiotherapy. Many improvements have been made to increase the benefit/risk ratio and broaden the type of tumours that can be treated with radiotherapy. However, these improvements have so far not been able to eliminate radiotoxicity. In the context of a pelvic tumour, when treating an organ in this region, which may be the prostate, a gynaecological organ, etc., several organs are in the radiation field and are therefore exposed to ionising radiation. Depending on the nature of the tissues making up the organ, some may be more radiosensitive than others. In the pelvic region, the digestive tract (small intestine, large intestine and rectum) is radiosensitive and can be exposed to ionising radiation. Exposure to these tissues results in radiation-induced damage. Tissue damage can vary in severity from patient to patient and, in the worst case, can affect the quality of life due to the disabling symptoms it can cause. In 2010, it was proposed to group radiation-induced gastrointestinal symptoms into a condition called Pelvic Radiation Disease (PRD).

The purpose of naming a condition was to quickly identify patients with gastrointestinal symptoms following pelvic radiotherapy and provide relief by putting a name to their symptoms. Today, the mechanism underlying the development of radiotoxicity is still unknown. Still, in recent years a new hypothesis concerning the involvement of a compartment of the intestine not previously considered has been identified: the gut microbiota. Indeed, the intestine shelters in its lumen tens of billions of microorganisms involved in numerous processes linked to the functionality of the digestive tract. As the gut microbiota is connected to the host and forms a mutualistic symbiosis, it is seen as a potential actor in the development of gut radiotoxicity. Several clinical studies have shown that patients have altered gut microbiota following pelvic radiotherapy, but the timing of the onset of this alteration is still unclear, as are its consequences. Our study aims to understand better when the breakdown of the mutualistic symbiosis occurs after exposure to ionising radiation and what the results might be for the tissue. Because there is still no curative therapy for patients suffering from pelvic irradiation and because the gut microbiota appears to be one of the components responsible for radiotoxicity, we propose to test the effect of faecal microbiota transfer (FMT) in a preventive or curative way to see if it can be considered as a candidate for the treatment of patients in the context of PRD.

STATE OF THE ART

1. Pelvic cancers

. Incidence rate (y-axis) for each highest incidence pelvic area cancers (x-axis) (3).

Radiotherapy is a medical treatment involving delivering high-energy particles to damage or kill cancer cells (4). To better understand the effects of ionising radiation on cells, it is important to recall their life cycle. The cell cycle is the process by which a cell divides into two and consists of different stages, whose names are: G1, S, G2, and M.

(5,6)

The first step of the life cycle of a cell is the interphase with the G1 stage that prepares the cell to initiate division, followed by the S stage, which consists of DNA synthesis and leads to the G2 stage, which is the organisation of all the genetic material copied from the S stage before the cell divides. Finally, the M stage, short for mitosis, where the cell divides all the duplicated genetic material into two, leading to the formation of two cells (5) (Figure .3). Mitosis itself consists of different phases in which chromosomes are replicated and divided to give rise to two cells with identical genetic material (7) (Figure .4).

The role of radiotherapy in the therapeutic strategy for pelvic cancers

A. Principle of radiotherapy (6).

Radiotherapy aims to eliminate cancer cells. The effectiveness of radiotherapy relies in part on its ability to induce damage to the DNA of cells directly or indirectly. Cancer cells do not function in the same way as normal cells. Because of the lack of signals, they cannot stop growing, dividing rapidly, staying together, specialising and having a functional repair system (8). The consequences of these dysfunctions are that cancer cells are more likely to be at any cell cycle stage, particularly at the G2-M. The last step seems to be when the cell is most vulnerable, which could be explained by the fact that it is the time when the cell contains the most DNA. Furthermore, when cancer cells are already struggling to fix potential damage, at the G2-M stage, the repair system does not have much time to orchestrate and be effective before the cell divide (9,10). Thus, cancer cells seem sensible for radiotherapy, but it is essential to consider normal cells alongside cancer cells. Some normal cells have a proliferative role, meaning they are also more likely to be in the G2-M phase.

The effect of ionising radiation can be direct or indirect. The first process involves the energy deposition to the DNA directly, causing single or double-strand breaks in the DNA. The second occurs when the radiation collides with the water molecule, which is the main component of the cell, inducing the formation of free radicals. The product of the radiolysis of water can act on DNA and damage it (11). The consequences of all radiation damage to cells can occur within hours of radiation exposure, in all organs exposed, with cells undergoing apoptosis, mitotic death, necrosis and senescence, a temporality that goes from day, weeks and even months later (12).

Radiotherapy can have many actions on cancer cells but also on healthy cells. Its use depends on the patient and the tumour to be treated, considering the therapeutic objective, the type of radiotherapy, the calculation of the total dose in a particular volume administered to the patient, and the protocol to be followed. A competent medical team will make all these decisions considering all the parameters necessary to provide the best treatment for the patient. As radiotherapy can affect healthy cells and tissues, it is crucial to consider this when developing treatment plans. The main objective is maintaining a positive balance between eliminating cancer cells and preserving healthy tissue.

Radiotherapy is an integral part of the treatment strategy for pelvic cancers. Depending on the patient and tumour characteristics, clinicians will choose the better treatment for the patient.

The use of radiotherapy in pelvic cancer is diverse and can therefore have different purposes, such as:

-Curative: it implies that this is the only treatment given to the patient (13).

-Neo-adjuvant: it aims to support other therapies (surgery, chemotherapy, etc.) to make them more effective.

-Adjuvant: it is used to reduce the risk of relapse after surgery.

-Palliative aims to relieve the patient when all other therapies have failed (14).

For each type of application, the clinician has different types of radiotherapy available.

The choice of the radiotherapy type must consider the organ affected as tumour characteristics.

In pelvic cancer, clinicians can use three types of radiotherapy. The first is external beam radiotherapy (EBRT); in this case, the source comes from an exterior machine. Next comes brachytherapy consisting of implanting a radioactive device inside the body. Finally, there is radiopharmaceutical, drugs containing radionuclides, and the patient takes them as medicine.

For EBRT treatment, the patient must go to a hospital or centre. Patients sit or lie down with the machine pointing at the target organ. The beams suitable and most common for this type of radiotherapy are most often photons (X-rays) followed by particles (mainly protons). The machine used to deliver the beams is specific to these beams. X-rays require a linear accelerator (for X-rays) (Figure. Different external beam techniques exist with the same objective: to enhance the tumour dose and the efficiency of radiotherapy. All these techniques require tumour imaging to prepare and calculate the treatment for each patient. All the methods mentioned below use both photon and proton beams. In photon EBRT, the generation of the beams is modulated using a collimator that directs the beams to the tumours, regardless of their shape. The main techniques are:

-Three-dimensional conformal radiation/proton therapy (3D-CRT, 3D-CPT): During the radiotherapy session, beams with the same intensity from different directions will reach the tumour so that it is fully affected (17). 3D-CPT use passive scattering beam delivery, which involves the broadening of a proton beam by a scatterer, which is then matched to the target using a collimator and a capacitor. This technique's limitation is controlling the dose distribution in the areas surrounding the tumour (18,19).

-Image-guided radiation/proton therapy (IGRT/IGPT): similar to 3D-CRT/3D-CPT but includes imaging techniques before or during each radiotherapy session, it allows the clinician to replace the patient for a better match. IGRT is the best run when the tumour is in a moving area (20).

-Intensive modulated radiation/proton therapy (IMRT/IMPT): close to 3D-CRT/3D-CPT except that all beams do not have the same intensity; each beam is divided into several beamlets with individual intensity levels. This facilitates the treatment of tumours with concave or complex shapes. This technique allows the delivery of a higher dose to the tumour (21). IMPT use Pencil Beams Scanning (PBS), a more recent approach where the proton beams are shaped with scanning magnets that allow them to reach the tumour layer by layer with different energy levels (18,19). There are several sorts of IMRT, for example, arc therapy, where a medical linear accelerator rotates around the patient during radiotherapy treatment. Among them is helical tomography (HT), or tomotherapy, a combination of EBRT and CT scan. It allows a scan to be taken before each radiotherapy session; the same machine will then deliver the radiotherapy to the tumour just after adjusting the treatment to the tumour seen on the scan (22). Like tomotherapy, volumetric modulated arc therapy (VMAT) is another arc therapy where the delivery radiotherapy beam is in an arc shape. During radiation treatment, the moving machine will change the shape and intensity of the beams. Due to some pelvic tumours' complex shape and location, they could not be easily accessible. In this case, HT and VMAT appear to be interesting alternatives, mainly to avoid the radiation exposure of the organs surrounding the tumour (23)(24)(25)(26)(27)(28).

EBRT techniques photon beams are still the most common. Nevertheless, in recent years, EBRT techniques using proton beams are attracting interest from clinicians. The advantage of protons is that their speed is reduced when they enter the matter, leading to their energy loss.

Proton therapy, therefore, appears to be an excellent option for delivering a dose to the tumour with minimal impact on the surrounding normal tissue. Proton therapy is not widely used in treating pelvic cancers in France, but it remains an interesting therapeutic option. The development of new techniques and research into their therapeutic value will perhaps lead to their more frequent use in specific cases of pelvic tumours (29)(30)(31).

Another type of EBRT is stereotactic irradiation (SRT) which includes stereotactic body irradiation (SBRT), stereotactic ablative body irradiation (SABR), or stereotactic body proton beam therapy (SBPT) when using proton instead of the photon, and stereotactic radiosurgery (SRS) (32). The principle of SRT differs from the conventional EBRT technique described above in that it delivers a high dose of radiation to a small local tumour using multiple small beams. Each beam is of low intensity, but all beams meet at the same point so that their target receives a high dose of radiation. In the case of SBRT, the radiation dose is given in several times, whereas in the case of SRS, it is given in one time (33). SRT allows patients for whom surgery is not an option or who have a small tumour volume to receive adequate treatment. The primary advantage of SRT is to decrease energy delivered to any normal tissue around the tumour. In addition, when the dose delivery is fractioned, the number of sessions is less than with conventional radiotherapy, which is more convenient for the patient. SRT is feasible with technologies such as Cyberknife and Truebeam for photon SBR and a proton therapy system for SBRT.

Some studies have worked on the alternative to other treatments with SBRT to boost radiotherapy for prostate or gynaecological cancers. SBRT seems to reduce the risk of developing radiation-induced complications, but protocols must be optimised and further studies must be conducted to ensure this (34)(35)(36)(37).

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) 38
EBRT is a widely used radiotherapy type in pelvic cancer, but needleless brachytherapy is also a common treatment.

The radioactive device is administered in brachytherapy to reach the patient's tumour locally.

The radioactive device is sealed or unsealed depending on the tumour's location. If they are sealed, they are contained in seeds, ribbons or capsules that the doctor places in the tumour (interstitial brachytherapy) or a cavity, either natural or created by surgery (intracavity brachytherapy), with a catheter or an applicator. If they are not sealed, the radioactive source is not placed in a container but is available in liquid form, administered orally or intravenously; they reach their target thanks to their biological and physicochemical properties. Different ii. Brachytherapy types of radiation can be emitted by radioactive sources, such as: a,b particles or g-rays. The most common sealed radioactive source used in pelvic cancers are iridium-192, cobalt-60, iodine-125, palladium-103 and celsium-137 (Table .1). The choice of the radioactive source depends on the type of brachytherapy the patient will receive. There are several types of brachytherapy, the most common being:

-Low-dose Rate (LDR) brachytherapy: Once implanted, it can remain in place for several days (temporary brachytherapy) or even forever (permanent brachytherapy).

-High-dose Rate (HDR) brachytherapy: The radioactive implant is placed for several minutes and removed. The treatment is repeated several times, and the patient has the choice of staying in the hospital for the treatment or coming in just to receive daily or weekly brachytherapy (39).

Table 1. Most common sealed radioactive source in brachytherapy for pelvic cancer (40) The advantage of short-lived radioactive source is that the patient does not need to remove them, requiring only one procedure. Brachytherapy has advantages concerning the patient's quality of life over EBRT, both in terms of treating the tumour with less risk of damage to surrounding normal organs (Figure .8) and in terms of comfort, as some patients do not have to make repeated visits to the hospital for their radiotherapy.

Brachytherapy is a valuable resource in treating pelvic cancer and can be applied as a single treatment or combined with chemotherapy, hormone therapy or surgery. It can also be used as a booster to treatment after EBRT alone or chemoradiotherapy with EBRT. uniformly; it depends on the location of the cell about the position of the radiopharmaceutical on the cell. For example, if the tumour cells are numerous, the radiopharmaceutical is more likely to eradicate them than if the number of tumour cells is small because, in this case, the radiopharmaceutical is less likely to deposit sufficient energy in the nucleus of the cell and thus kill it (Figure.9). However, by changing the activity of the radiopharmaceutical, it is possible to increase its chances of killing even small numbers of cancer cells, at the same time spare the normal surrounding tissue. The added advantage of radiopharmaceuticals is that they can be used in cases where the tumour cells are spreading over the body, EBRT would not be able to target the cancer cell in this case, but as radiopharmaceuticals bind to a specific receptor in the cell, they can have more precise action. Also, for a theragnostic approach, clinicians can use the same compound for both imaging and treatment; the imaging version of the compound iii. Radiopharmaceuticals or internal vectorised radiotherapy allows clinicians to know whether the radiopharmaceutical is targeting the desired cell and, accordingly, is effective in killing it (42).

The use of radiopharmaceuticals for pelvic cancer is mainly for prostate cancer, but some studies have investigated whether they could also be beneficial for gynaecological cancers.

There are several types of radiopharmaceuticals, including radionuclides alone or coupled to other atoms (small molecules), peptides or antibodies. Radionuclides can also be incorporated into nanoconstructs, but these have only been tested pre-clinically, and finally, they can be incorporated into microspheres. Most radionuclides in radiopharmaceuticals emit particles a or b. For pelvic cancer, in particular prostate cancer, only small molecules (radium-223 dichloride or Lu-PSMA-617) have been approved, and yttrium-90 microspheres are under investigation as the combination of radiopharmaceutical with other cancer treatment (42,43).

Radiotherapy is a powerful treatment for cancer treatment. The fact that it can be delivered using many different techniques means it offers the clinician many choices. The radiotherapy protocol should be based on the type of radiotherapy used to treat the patient. Another part of preparing for radiotherapy treatment is choosing the total dose needed to be effective and its distribution.

The delivered radiation dose is the energy deposited by ionising radiation as it passes through a matter. The total dose is chosen according to the dose that will have the desired effect on the tumour, while the delivery protocol takes into account healthy tissue and depends on the type of organ surrounding the tumour. Some organs are considered at risk (OAR) according to specific characteristics. First, the structural organisation of the organs can have a serial, parallel or serial-parallel function architecture. In serial architecture, all the organs depend on each section that makes them up.

c. Dose total and dose distribution

Thus, when organs with a serial structure are irradiated, even a small part can cause organ functionality problems. In parallel architecture, the sections of the organ work independently.

Therefore, when an organ with a parallel structure is irradiated, the volume of the irradiated organ must be large for it to cause functionality problems. Concerning serial-parallel structure, organ toxicity comes from irradiated volume and total dose received (44).

Proliferation capacity must also be considered. Tissues with high proliferative capacity are more likely to develop acute toxicity, while tissues with low proliferative capacity are more likely to develop late toxicity (45).

Although the OAR are not necessarily the direct target of irradiation, it can be exposed to radiation and, despite receiving a lower dose than the tumour, is likely to create damage to the normal cells. Cells exposed to radiation can release signals reaching neighbouring cells, which will respond in different ways ranging from changes in gene expression to cell death. Thus, radiation can have a bystander effect that contributes to radiation-induced toxicity.

The establishment of OAR allows a reflection above the total dose to administrate and distribute with objectives to spare as possible OAR and avoid radiotherapy normal tissue toxicity.

Thus, some models have been developed to measure the tumour's and surrounding organs' is that it can quickly estimate a tumour's sensitivity to dose per fraction. Thus, if the ratio is i. Concept of organ at risk low, the sensitivity to the dose per fraction is high. On the contrary, the sensitivity to the dose per fraction is low if it is high. This tool allows clinicians to orientate therapeutic choices that are favourable to hypofractionation. For example, this is the case for prostate or breast cancer with a low a/b ratio, which would thus be more sensitive to higher doses per fraction (46).

Algorithms also have a role to play. Indeed, Monte Carlo simulation has significantly improved the treatment plan. It allows clinicians to see the distribution of particles in the matter and therefore the dose distribution in the tissue (47). This information, combined with that provided by the linear-quadratic model, can help the clinician calculate the therapeutic index and develop a treatment plan with the best benefit/risk ratio.

The therapeutic index considers the tumours' response to radiation and the late response of normal tissues to select a protocol that will kill the maximum number of cancer cells with the minimum risk of developing late toxicity. Late toxicity in normal tissue is regarded as the worst for the patient as it is considered permanent (48).

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) 48
An OAR can still be exposed to ionising radiation because it is able to tolerate a certain dose before developing toxicity: this is the notion of tolerance dose.

Tissue tolerance to a certain dose has been calculated on the basis of clinical data, which has made it possible to obtain tools such as the TD (tolerance dose)5/5 or 50/50, which represents the radiation dose for which there is a 5% or 50% probability of developing toxicity in the next 5 years. Also, the introduction in the 1980s of the dose-volume histogram (DVH), which gives

ii. The notion of dose tolerance Figure 10.Estimating the dose where cancer cells will be damaged the most and normal tissue the least (48) the distribution of a dose in a tissue helped to provide data for identifying doses that should not be exceeded. Thus, it was subsequently established that for each organ deemed at risk, a dose was not to be exceeded depending on the type of radiotherapy used. Therefore, when a clinician establishes a radiotherapy protocol based on tumour and patient parameters, he should calculate the dose distribution using a DVH and, depending on the results, rely on the known tolerance dose for the tissue to adapt his protocol.

He can also use a mathematical model such as the Normal Tissue Complication Probability which uses different information based on the literature, the patient, his tumour, the clinician's planned protocol, and the DVH, to calculate the probability of developing toxicity and thus adapt his protocol.

For cancers of the pelvic region, several organs have been identified as being at risk, and a dose tolerance has been established for each of them (Figure.11). In this area the most identified organs at risk in the digestive tract are the small intestine and the rectum. Nevertheless, in a study on a patient with prostate cancer who developed late radiotherapy-induced lower intestinal toxicity, the dose received to sigmoid has been correlated to grade 1 diarrhoea. To avoid grade 1 diarrhoea during IMRT radiotherapy for prostate cancer, authors proposed to limit the volume of sigmoid receiving 40Gy or 30Gy respectively to 10 and 16% (49). Another study identified the colon as OAR for IMRT in prostate cancer and had dose constraints to not cross for this organ (50). (51). (51) Identifying the dose to be given to the patient is only one part of establishing the radiation therapy protocol. Drawing up a scheme for administering this total dose is also necessary. As mentioned above, cancerous cells have poor repair capacity compared to normal cells. Delivery of fractioned dose irradiation is allowing time for normal cells to try finishing the repairing process without letting enough time for tumorous cells to do it.

Based on the characteristics of the tumour, its location and the patient profile, and thanks to the tools available to clinicians, a total radiation dose will be selected, and a protocol for distributing this dose will be implemented. Today there are several types of protocols for delivering radiotherapy: conventional fractionation, hypofractionation and hyperfractionation, accelerated radiotherapy fractionation and palliative treatment fractionation.

-Conventional fractionation: most common radiotherapy protocol. Doses from 1.8 to 2.0 Gy are delivered daily for 5 days for 6 or 7 weeks.

-Hypofractionation: the dose delivered is higher than conventional fractionation (2.5-7 Gy), and the treatment is given once a day or less frequently. The total duration of treatment is shorter than with conventional fractionation.

-Hyperfractionation: lower dose than conventional radiotherapy (1.1-1.2 Gy) but given twice daily. The total treatment time remains the same as for conventional fractionated radiotherapy.

-Accelerated fractionation: the total dose is delivered in less time (with delivery dose per fraction between 1.6-1.8 Gy) than conventional radiotherapy, and the total treatment time is also shorter.

-Palliative treatment fraction: this can be given when the cancer is considered incurable. The first fraction is a high dose of 3 to 4 Gy and subsequent fractions are lower (2 Gy per fraction) [START_REF] Mc | Towards clinical translation of FLASH radiotherapy[END_REF].

Research into radiotherapy and how to deliver it to achieve maximum tumour control and less damage to normal tissue is still active. Recently, the FLASH (ultra-high dose rate of radiation in a very short time (>40 Gy/s) treatment protocol has been proposed as having an interesting benefit/risk balance compared to conventional dose rate radiotherapy treatment plans (53).

However, this technique is not yet used in clinics.

iii. Fractionated radiotherapy protocol

Despite clinicians' best efforts to spare normal tissues, some still receive a certain dose, leading to complications that can occur during and after radiotherapy.

It should be considered that the number of patients surviving their cancer is increasing due to better patient management, which will lead to an increase in the number of patients suffering from radiation-induced toxicity. A 2020 study calculated the 2030 incidence of pelvic radiotherapy-induced complications from the incidence rate data at that time to be 5-10% [START_REF] Bryant | Trends in Radiation Therapy among Cancer Survivors in the United States, 2000-2030[END_REF].

In 2014, the incidence of complications induced by abdominal and pelvic radiotherapy was higher than that of inflammatory bowel disease (IBD) [START_REF] Hauer-Jensen | Radiation enteropathy-pathogenesis, treatment and prevention[END_REF]. Therefore, it is essential to define these complications and their occurring risk.

The PRD was defined in 2010 by Andreyev and his team as "transient or longer-term problems, ranging from mild to very severe, occurring in non-cancerous tissues following radiotherapy treatment of a tumour of pelvic origin". In addition, they state that all patients describing symptoms in the following organs: bowel, urinary tract, sexual organs, bone, or skin after pelvic radiotherapy may have developed PRD. The goal of defining PRD was urgent because many patients were in treatment drift or had been diagnosed with Crohn's disease (as were 50%

of patients with PRD in 2010) [START_REF] Andreyev | Defining pelvicradiation disease for the survivorship era[END_REF]. This could be due to the heterogeneity of symptoms in PRD, many organs can be involved, and until now no pathology was referenced for the enumeration of symptoms. In addition, some patients were not warned of the potential effects of their radiotherapy treatment, so they could not use this information to make the connection and alert the clinician to the onset of symptoms. In other cases, some patients may have been frightened into thinking it was a recurrence of their cancer and did not mention it to their doctor.

Moreover, some symptoms may be difficult to talk about, as people may consider them shameful. Without a defined pathology, it is more difficult for clinicians and patients to know what symptoms may occur after pelvic radiotherapy. Also, putting patients' symptoms into words can reassure them.

PRD affects a large number of patients, 90% who have undergone pelvic radiotherapy report a change in bowel habits and is responsible for a moderate to a significant decrease in quality of life for 20 to 40% of patients [START_REF] Andreyev | Gastrointestinal symptoms after pelvic radiotherapy: a new understanding to improve management of symptomatic patients[END_REF]. The PRD may occur during or 3 months after the end of

The Pelvic Radiation Disease (PRD)

radiotherapy, this is called acute PRD and is generally transient. Also, it can also occur from 3 month after the end of radiotherapy of patients who have undergone pelvic radiotherapy and is defined as a chronic condition. [START_REF] Andreyev | Gastrointestinal problems after pelvic radiotherapy: the past, the present and the future[END_REF]. The relationship between acute and late PRD is still unclear today. Some proclaim there is a consequential effect of radiation, so the more acute PRD is pronounced, the more the patient risks developing late PRD with the same severity [START_REF] Dörr | Consequential late effects in normal tissues[END_REF]. But this is not consensus standing.

The symptoms of PRD can have different degrees of severity. Certain scores and indices have been developed to help clinicians determine the severity of a patient's injury. Some measure the severity of symptoms in each organ after the administration of an anti-cancer drug (Common terminology criteria for adverse event or CTCAE), detailing different grades for each symptom [START_REF]Common Terminology Criteria for Adverse Events (CTCAE) | Protocol Development[END_REF], others after irradiation (Toxicity criteria of the Radiation Therapy Oncology Group or RTOG) (Figure 11). The classification ranges from 1, where the injury is the least severe, to 5, which corresponds to the patient's death. These classifications provide a measure of the acuity of the PRD. [START_REF] Chinnachamy | Evaluation of Interobserver and Interscale Agreement in Assessing Late Bowel Toxicity after Pelvic Radiation in Patients with Carcinoma of the Cervix[END_REF][START_REF] Parente | Clinic, Endoscopic and Histological Features in Patients Treated with ICI Developing GI Toxicity: Some News and Reappraisal from a Mono-Institutional Experience[END_REF] Each phase of PRD has common or specific symptoms. In the acute phase, possible symptoms include nausea, diarrhoea, tenesmus, abdominal cramps, mucus discharge, an urgency to defecate, loss of appetite and bleeding. In chronic phase of PRD, the same symptoms are present, but with the added risk of complications such as bowel obstruction [START_REF] Morris | Pelvic radiation therapy: Between delight and disaster[END_REF]. Symptoms of PRD can be explained by events occurring inside the organs. The colon is not an organ targeted directly by pelvic radiotherapy. However, as discussed above, the targeted organ is never the only one exposed to ionising radiation. Although neglected in identifying non-target organs but still exposed during pelvic radiotherapy, some studies consider the colon as likely to be in the radiation field and therefore susceptible to radiation-induced toxicity [START_REF] Jadon | A systematic review of dose-volume predictors and constraints for late bowel toxicity following pelvic radiotherapy[END_REF].

A. Symptoms of PRD

The colon is the last fraction of the intestine and is divided into two sections, from the closest to the small intestine to the closest to the rectum: the proximal colon and the distal colon. In the process of digestion, the events that occur in the colon are the last stage where the product of digestion is composed only of water and indigestible matter, here water and salt are absorbed giving the stool its consistency. During this phase, mucus is produced to facilitate the passage of the consistent stool into the rectum. The structure of the colon is organised to allow this process.

The tissue of all the digestive organs within the colon is organised similarly with four layers.

From the inside out, there is the mucosa, submucosa, muscle layers and serosa (Figure 12.).

The mucosa is protected by two layers of mucus: the inner and outer. The inner mucus layer is the one closest to the epithelium while the outer mucus layer is the furthest away. Bacteria can penetrate the outer mucus layer but not the inner mucus layer. The mucus acts as a barrier to prevent direct contact between the luminal contents of the gut and the epithelial cells. Mucus prevents dehydration of the epithelial cells or their exposure to a potential pathogen but allows the passage of molecules beneficial to them (65) (Figure 17.).

Host injuries in PRD

A. Structure of the colon (large intestine) exposure including radiation, their position at the base allows them to be as far away as possible from the intestinal lumen, ensuring their protection. Quiescent stem cells are less sensitive than active stem cells (due to their quiescence) and have been given a role in response to oxidative stress. The role of the stem cell is to maintain the stem cell population and to participate in the renewal of the epithelium by producing transit amplifier cells.

Transit amplifier cells are intermediate between undifferentiated and differentiated cells. These cells can give rise to any type of differentiated cell of the colonic epithelium. These cells have a high proliferative capacity and therefore participate in epithelium renewal by replacing dead differentiated cells.

Finally, the higher the cell is in the crypt, the higher its differentiation level. Apart from the deep crypt secretory cells, differentiated cells are found at the base of the crypt and participate in support of colonic stem cells. At the top of the crypt are only differentiated cells, including cells specialised in absorption (enterocytes) or secretion (goblets, enteroendocrine and tufts cells). They all have a common structure, namely the microvilli in the apical part of the cell.

The organisation of the microvilli increases the exchange surface with the intestinal lumen.

-Enterocytes: these are cylindrical cells allowing the exchange of nutrients with the intestinal lumen. (Figure 13.A.).

-Goblets cell: These cells have a vase-like appearance with the apical pole larger than the basal. Apically, there is a secretory vesicle full of mucin, a glycosylated protein which, when secreted, forms the mucus found at the top of the colonic epithelium (Figure 13.B).

-Enteroendocrine cells: these cells have the shape of an Erlenmeyer flask, they produce peptides and hormones that are released to the blood (endocrine way), to around the environment (paracrine) or to the nervous system. Their product is formed to answer received signals (Figure 13.C.).

-Tuft cells: they are characteristically oval and owe their name to the brush-like appearance of their apical pole. They are chemoreceptor cells involved in inflammation and repair. They belong to the colonic stem cell niche. In the intestine, an experimental study of helminth infection in mice showed that their production of IL-25 cytokines led to the elimination of the helminth. During this infection, their numbers are increased, which confirms their involvement in the elimination of the helminth (67) (Figure 13.D.).

All these cells contain proteins whose function is to protect them from molecules or to allow communication between the exterior and between them. At the apical level, there are zonula occludens which are tight junctions, a barrier to certain molecules in the intestinal lumen.

Below the zonula occludens is the zonula adherens, which is an adhesive junction in perpetual motion, assembled or disassembled, it responds to mechanical or biochemical signals and to structural changes in its microenvironment. At the lateral levels are the macula adherens (desmosomes) and the macula communicans (gap junction). The desmosome holds the cells together to participate in the structure of the tissue, it also transmits signals between the cells.

The gap junction allows a connection between the cytoplasm of two cells leading to the transmission of signals (Figure 13.A.).

(68-71)

It is a connective tissue that lies under the epithelium and comprises vessels immune cells and exocrine glands. The number of cells populate lamina propria notably fibroblast, immune and neural cells.

-Fibroblast: These cells, with their star shape, are supporting cells. They help renew the extracellular matrix by secreting proteins and glycoproteins, its main components. They can also produce and destroy collagen. In addition, they have an anti-infectious role.

-Immune cells: There is different types of immune cells in the lamina propria, all of which are part of the response to a toxic event, particularly as part of the inflammatory response. They have the ability to produce molecules that allow communication with ii. Lamina propria: C. example of an enteroendocrine cell with the process of GLP1 production and its destination [START_REF] Cummings | Gastrointestinal regulation of food intake [Internet[END_REF]. D. Tuft cell with all its constituents and how it is positioned in relation to the neighbouring cell [START_REF] Hendel | Tuft Cells and Their Role in Intestinal Diseases[END_REF].

their environment. Each immune cell comprises several types with different phenotypes; a non-exhaustive description is given in the following paragraph.

Neutrophils:

They are part of the polymorphonuclear cells such as polynuclear basophils and eosinophils.

They are called polynuclear because of their polylobed nuclei. Neutrophils are the most abundant circulating immune cells (65% of leukocytes in the blood). In the blood, they do not remain for more than 24 hours, and once in the tissues, their life span is one to three days.

Neutrophils acquire their phagocytosis function in the tissue. They also can produce messenger molecules and signals that are received by cells within their environment. They can have a proor anti-inflammatory role depending on the type of environment in which they are found. In case of inflammation, neutrophils recruit other leukocytes and produce a recognisable molecule that induces their apoptosis or phagocytosis by macrophages.

Macrophages:

Part of the myeloid lineage, it is a cell whose main action is phagocytosis. Macrophages are only found in tissues, but a precursor form is found in the blood, called monocytes.

Macrophages in tissues are called resident macrophages. In some tissues, the pool of macrophages is maintained by self-renewal, but in others, such as the gut, the resident macrophages are mainly derived from circulating monocytes. It seems that the intestine is the organ where resident macrophages are most numerous; this is due to the constant contact with the elements of microorganisms living in the intestinal lumen and all the molecules coming from the food etc... Macrophages have various functions depending on the environment in which they are found. The three populations that best describe the main function of Mast cells: They have the particularity of containing a lot of heparin or histamine granules in their cytoplasm which has led to confusion in distinguishing them from basophils for a while.

They have a role in inflammation and repair mechanisms. In a mouse and human model, two subtypes have been identified according to the type of proteoglycan constituting their granules.

The distinction can be made by staining specifically for one type of granule. The name of the mast cells depends on the model. For example, in the mouse model, they are called connective mast cells or mucosal mast cells, whereas, in the human model, they are called tryptase mast cells and chymase/tryptase mast cells. In Humans, their names depend on the constituents of their granules. Thus, the tryptase mast cell has granules containing tryptase, whereas the NKT can recruit all types of immune cells by producing IFNg, IL-2 and IL-4 and GM-CSF and TNF. Some lymphocytes can have both cytotoxic and non-cytotoxic action: LTregs. Their name corresponds to their regulatory action, whereas all other T lymphocytes are effectors.

LTregs have a broad spectrum of action. They are distinguished according to their expression, The serosa is composed of dense conjunctive tissue with many adipocytes and vessels. At the base of this layer is the mesothelium, a visceral leaflet of the peritoneum.

Exposure to ionising radiation of colonic tissue will influence its organisation but also on its function.

Generally, radiation exposures have consequences on tissue that could occur directly to several years after exposition which can be resumed in the following figure (Figure .18).

(

) 48
Colon epithelial cells are radiosensitive. Some of them can have a high proliferation rate leading their DNA to be more prone to radiation-induced toxicity. A few hours after radiation exposure, some epithelial cells may undergo apoptosis mediated by the p53 protein [START_REF] Merritt | The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice[END_REF].

Radiation exposure to colonic stem cells causes abnormal mitosis in some of them [START_REF] Hua | Distinct Levels of Radioresistance in Lgr5+ Colonic Epithelial Stem Cells versus Lgr5+ Small Intestinal Stem Cells[END_REF], leading d. Serosa

B. Consequences of radiation exposure to the digestive system a. Endothelial and epithelial permeability Figure 18. Detailed event could happen on tissue after exposure to ionizing radiation (43).

to a mitotic catastrophe that can occur one to several days after exposure. Moreover, epithelial cell death is not the only consequence of radiotherapy. Even if the cells do not die, they may lose proteins forming tight junctions [START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF]. Therefore, exposure of the colon to radiation increases tissue permeability (1). The mechanism of repair and proliferation of cells that have resisted to radiation exposure depends on the endothelial cells that are also affected. After exposure to colorectal irradiation, it has been shown that the expression of ICAM-1, which is an adhesion marker, increases. This has been linked to increased expression of TNF and IL-1 leading to leukocyte extravasation from the endothelium into the epithelium by stimulating endothelial cells [START_REF] Ikeda | Expression of ICAM-1 and acute inflammatory cell infiltration in the early phase of radiation colitis in rats[END_REF]. In conclusion, it appears that tissue and endothelial injuries lead to the recruitment of the immune system to limit the damage. This could explain the increase in leukocyte extravasation into the tissue. Thus, exposure to irradiation leads to the establishment of an inflammation process. Inflammation occurring in the tissues should have protective action leading to reparation of tissue, but its persistence over time seems to be deleterious and can contribute to the stimulation of fibroblasts in the submucosa leading to the initiation of fibrosis mechanisms, an excessive and pathological scaring [START_REF] Mintet | Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis[END_REF].

Inflammation can be triggered by danger-associated molecular patterns (DAMPs) or by activating pathogen-associated molecular patterns (PAMPs). Inflammation triggered by the activation of DAMPs only is called sterile inflammation. In the case of radiotherapy, inflammation can be triggered by the death of epithelial and/or endothelial cells. In addition, DAMPs, as PAMPs, can activate non-immune cells such as endothelial cells, epithelial cells and fibroblasts. Thus, in the colon, immune cells residing in the colonic tissue, epithelial cells, endothelial cells and fibroblasts can activate the recruitment of other cells, leading to inflammation.

Neutrophils:

They are recruited when there is a danger to the tissue. After irradiation, it seems normal to have a certain number of neutrophils where the tissue is damaged. However, their presence can last and, in this case, lead to a process of hyperpermeability [START_REF] Panés | Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats[END_REF]. Neutrophils and the consequences of their recruitment were measured in a study where animals received 10Gy abdominal irradiation. After irradiation animals received tepoxalin (an inhibitor of proinflammatory enzymes such as cyclooxygenase and lipoxygenase) by intragastric b. Sterile inflammation administration. To observe the effect of tepoxalin, they, therefore, measured leukotriene B4 (neutrophil chemoattractant) levels in tepoxalin-treated and untreated animals 2 hours and 4 hours after irradiation. The two groups had different amounts of leukotriene B4 and MPO (leukotriene B4: 30pg/mL for treated animals and 280pg/mL for untreated animals, MPO: 0.85 +/-0.2 for untreated rat and 0.49 +/-0.1 for treated rat). The decrease in leukotriene B4 and MPO was correlated with the decrease in tissue damage, supporting the hypothesis important primary neutrophil recruitment can be deleterious for the tissue [START_REF] Panés | Tepoxalin inhibits inflammation and microvascular dysfunction induced by abdominal irradiation in rats[END_REF].

Neutrophils are not the only immune cell present and involve in sterile inflammation.

Macrophages:

In the context of colorectal irradiation, a study shows that the polarisation of M1 macrophages into M2 macrophages induced by a liposaccharide (LPS) or flagellin could improve the condition of the animals. This implies that in a damaged tissue state, M1 macrophages are present. In addition, the study focuses on the production of IL-10, which induces polarisation from M1 to M2 via IL-10 increase. The increase in IL-10 has been correlated with epithelial cell proliferation and regeneration. M2 macrophages could also produce IL-10. Thus, in the context of a deleterious inflammation process caused by irradiation, it would seem that M1 is dominant and that the transition to an M2 phenotype would allow the epithelium to regenerate [START_REF] Lacavé-Lapalun | Flagellin or lipopolysaccharide treatment modified macrophage populations after colorectal radiation of rats[END_REF].

Mast cells also have been described in the context of radiation-induced sterile inflammation.

Mast cells:

A study examined mast cells' role in inflammation generated by exposure of the colon to ionising radiation. To do that, they exposed c-KIT-/-animals, who had the inability to produce mast cells, to a single dose of colorectal irradiation. They did some analysed in the colon and rectum 2 and 14 weeks after irradiation. They found that the genetically modified animals had the least amount of damage, implying that mast cells may play a detrimental role in the response of colonic tissue to irradiation. They then exposed colonic smooth muscle cell lines to high levels of chymase and tryptase (proteins produced by mast cells). The consequence of this exposure was the proliferation of colonic smooth muscle cells. Thus, the conclusion of this study is that mast cells seem to be involved in the development of lesions of the epithelium.

Mast cells seem also involved in the dystrophy of muscularis mucosa via activation of this recruitment, proliferation and survival by smooth muscle cells [START_REF] Blirando | Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice[END_REF].

Adaptative cells well also studied in the context of colorectal irradiation.

Lymphocytes:

The lymphoid cell population studied in colorectal irradiation are LTCD4 cells, particularly TH17 cells and LTreg.

Concerning TH17 cells, a study was carried out on their level after irradiation. After colorectal irradiation in rats, they showed that the TH17 population and the cytokines IL-6 and IL-1b increased. A hypothesis correlating this increase in TH17 cells, and these two cytokines was put forward. This study, therefore, concluded that the excess population of TH17 after colorectal irradiation was involved in the formation and/or maintenance of radiation-induced colonic lesions. This study also examined the LTreg population, which they measured in mesenteric lymph nodes in controls and irradiated animals. They found that this population increased after irradiation, but when they examined whether the relative expression level (in the mesenteric lymph node) of some of the cytokines produced by LTregs was also increased, they found that this was not the case, particularly for IL-10. ( 84). This implies that despite their increase, the LTregs could change their phenotype to one that may no longer be able to regulate the immune cell population.

This assumption was supported by in vitro results. The researchers co-cultured non-irradiated or irradiated iTregs with LTCD8 cells and assessed the ability of iTregs to inhibit the growth of LTCD8 cells. They showed that the LTCD8 population increases in the presence of irradiated iTregs compared to results obtained in the presence of unirradiated cells. This result means that irradiated iTregs seem to lose their ability to inhibit the LTCD8 proliferation [START_REF] Beauford | Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells[END_REF].

Immune cells are part of an integrated and extremely complex system that seems specific in each organ in which they intervene. Knowledge about each of these cells is increasing yearly, leading to a renewal of previously established hypotheses. In the context of PRD, the literature is not extensive, making it difficult to confirm that these immune cells are involved in toxicity or repair processes. Moreover, today the colonic ecosystem can be considered as an indissociable whole integrating the host and the billions of microorganisms populating the lumen of the intestine which have been named intestinal microbiota. Integrating the microbiota into the PRD equation adds a layer of complexity and the prospect of better understanding and perhaps discovering a new mechanism.

II. The microbiota

The microbiota refers to a consortium of microorganisms (bacteria, fungi, viruses, protists, and archaea) living in a specific environment (microbiome). In humans, the microbiota is found in every mucosal surface. As their composition depends on the microbiome, each microbiota is unique. All we know about it now is the fruit of a millennium of uses and studies.

Like any scientific discipline, microbiology has progressed in parallel with technological improvements. Its interdisciplinarity has contributed to developing concepts of primary importance in reflecting on its involvement in the environment and human biology.

For thousands of years, microorganisms have been used without people being aware, for example, in producing bread and alcohol through fermentation processes. Marcus Terrentius Varo was the first to identify invisible living beings in the marshes. He called them animalcule, a name that endured until 1889 when the name microbe (from the Greek, μικρός, "small and βίος "life") proposed by the surgeon Charles Sédillot was preferred. It was not until several centuries later (17th century) that these microbes would be physically identified thanks to the invention of the microscope by Athanius Kirsher and its refinement by Antoni Van Leeuwenhoek. A new advance, the cultivation of microorganisms, carried out by Lazzaro Spallanzi, enabled a better understanding of their dynamics, notably by refuting the theory of spontaneous generation. After Lazzaro's work, when microbiology was confined to the field of the environment and human health, Louis Pasteur's work on pasteurisation boosted the popularity of this discipline by transposing it to the industrial field. However, at that time, the idea that microorganisms were anti-hygienic and pathogenic for humans and should therefore be eliminated was dominant. 3). A. Barnacles living on whales are an example of a commensal relationship that benefits only the barnacles, with the whales providing habitat and nutrients without receiving any benefit in return [START_REF] Natgeouk | Why Whale Barnacles Can't Keep a Secret and More Breakthroughs[END_REF]. B. Mutualistic relationship between buffalo and red-billed woodpecker. The bird feeds on the buffalo's parasite and warns the buffalo of the predator's approach by flying or calling [START_REF] Nick | 5 Greatest Symbiotic Relationships in Nature -SunnyScope[END_REF]. C. A mutualistic relationship called a lichen, which involves algae or cyanobacteria providing elements from photosynthesis to a fungus, while these fungi protect them from the environment [START_REF] Carylsue | Symbiotic Relationships in Marine Ecosystems[END_REF]. D. In the mutualistic relationship between the clownfish and the anemone, both are sources of nutrients for the other and protect each other from their respective predators (89). E. The cactus mistletoe is a parasite of the cactus, it benefits from the nutrients given by the cactus, while infection of the cactus by Tristerix aphyllus results in a decrease in its reproductive rate and photosynthesis (90). F. The parasite Ophiocordyceps unilateralis controls the infected ants and forces them to move to a place where the humidity is favourable for the growth of the fungus, and then the ants wait to die while the fungus grows and produces spores that can infect other ants [START_REF]How a parasitic fungus turns ants into 'zombies[END_REF]. of living together for certain organisms and so understand dynamics between living organisms.

In the medical field, the symbiosis between microorganisms and humans was established in the late 19th and early 20th centuries with the isolation of specific strains that were found to be directly related to human health. One of the first to establish a link between specific bacteria and human health was Henri Tissier, who observed a different degree of colonisation of Bifidobacterium in breastfed children compared to those fed infant milk. He attributes to Bifidobacterium, which is most abundant in breastfed infants, the ability to reduce the risk of developing diarrhoea observed more frequently in babies receiving infant milk. The same reasoning is found in Ilitch Metchnikov's evidence of the positive effect that lactic acidproducing bacteria could have on gastrointestinal disorders and the life span of the elderly. His hypothesis was based on the observation that Bulgarian people who habitually consumed yoghurt, curdled milk or kefir were found to have a significant healthy life expectancy. He also noted that a specific bacterium: Bacterium bulgaricus would have positive effects on the digestive system and immunity. Ilith Metchnikov then confirmed that certain microorganisms could have beneficial effects on humans.

The 19th and 20th centuries were marked by the loss of the previously negative image of microorganisms. The next revolution came at the end of the 20th century with the invention of sequencing and the project to sequence the human genome. It is important to remember that these sequencing techniques have revolutionised our vision of these microorganisms and the fundamental role they can play at different levels of human health in the evolution of diseases and even in response to treatment, especially for cancer.

The Sanger method was the first practical method for sequencing DNA fragments. It consists of extracting the DNA fragments from the cells, amplifying them by a polymerase chain reaction (PCR) and starting the sequencing process. The sequencing step involves the presence of a template DNA fragment, primers specific to the DNA fragment of interest, DNA polymerase, four types of deoxynucleotides (dATP, dGTP, dCTP, dTTP) and four types of dideoxynucleotides (ddATP, ddGTP, ddCTP, ddTTP). Each primer is positioned on the complementary side of the template DNA fragment, and then the DNA polymerase is used to attach the deoxynucleotide to the primer. Sometimes it is not a deoxynucleotide that binds to the elongated primer fragment but a dideoxynucleotide, i.e. a deoxynucleotide that lacks a 3'-OH, which results in an impossible linkage to a subsequent deoxynucleotide, so that the DNA polymerase stops elongating the fragment. Each dideoxynucleotide is coupled to a fluorescent molecule specific to one type of dideoxynucleotide (so there are four types of fluorescent molecules emitting at different wavelengths). To work, the sequencing step must have a concentration of deoxynucleotides 100 times higher than that of the dideoxynucleotides, which allows the elongation of each strand to occur and not always be stopped at the same point by the dideoxynucleotides. Once the sequencing step is complete, each synthesised fragment is electrophoretically separated according to the length of each fragment. The distance travelled by each fragment on the electrophoresis gel indicates the size of the fragment (the shorter the fragment, the faster and further it migrates). While migrating on the gel, each fragment encounters a laser that excites the fluorochrome on each dideoxynucleotide. The fluorescence emitted by the excited fluorochrome is received by a detector which transmits the signal, which is then compiled on a computer and analysed by specific software. In this way, the entire template DNA fragment can be reconstructed by sorting the fragments by length (the shortest being closest to the primer, the beginning of the fragment, and the longest at the end) and noting the specific nucleotide that ends each fragment (Figure 20).

A new vision of microorganisms

A. Sanger method [START_REF] Shrestha | DNA Sequencing -Sanger Sequencing Method • Microbe Online[END_REF] Using Sanger methods, the DNA of cultured bacteria was sequenced, and a specific gene attracted the researchers, 16S ribosomal RNA (16S rDNA), which is the gene coding for ribosomal RNA that forms the 16S subunit of the ribosome of prokaryotes. At that time, Carl Woese was looking for common genes between species to classify them. He and George Fox focused on ribosomal RNA and discovered the gene coding for 16S rDNA. This gene has the particularity of having conserved regions because they evolve slowly and are common to all prokaryotes. Still, it also has regions that evolve very quickly and are, therefore, extremely variable between different bacteria, which makes it possible to classify each bacterium in the prokaryotic kingdom. For example, a region has been found that allows bacteria to be identified simply by amplifying a fragment of their DNA in samples and sequencing them. After the Great Human Genome Project, which consisted of sequencing the entire human genome using Sanger technology from 1990 to 2003, some microbiologists became interested in applying this technic to microorganisms. The Sanger method is highly meticulous and allows only one specific fragment to be sequenced at a time, making sequencing time-consuming and highly accurate. In the 21st century, sequencing techniques have improved and have made it possible to sequence not only fragment by fragment, but all the fragments contained in the sample simultaneously, known as next-generation sequencing (NGS). These technologies have saved considerable time and reduced the cost of sequencing. For example, the human genome project took 13 years and cost $3 billion; in 2016, sequencing the human genome with NGS technology took three days and cost $1000. This is another significant step forward for genomic studies and, thus, for metagenomic studies (when studies are conducted on different species in an environment). It gave scientists, especially microbiologists, easy access to sequencing. Thanks to this advance, projects such as the Global Ocean Sampling, comprising samples from different oceans, have been sequenced to identify the microbial communities living in these different environments [START_REF] Rusch | The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific[END_REF]. Similarly, later on, the Human Microbiome Project, an American project was born, which focuses on sampling healthy people in five different parts of the human body (intestinal tract, mouth, skin, nasal cavity and vagina) where microbiota is found. The first part of the project was to identify and characterise the human microbiota, and the second was to determine the effect of the microbiota on health and disease [START_REF]Human Microbiome Project[END_REF]. In Europe, Metagenomic of the Human Intestinal Tract (MetaHit) began at the same time that the Human Microbiome Project and focused mainly on identifying microbial genes in health and disease (IBD and obesity) [START_REF][END_REF]. These three projects are metagenomic as they sequenced genes of different species in a given environment. Next Generation Sequencing or NGS is a generic term that includes all sequencing techniques that allow massive and parallel sequencing of the whole genome or fragments of DNA or RNA. Different NGS technologies are available depending on what needs to be identified and the degree of accuracy desired.

Today, several platforms exist to sequence DNA or RNA. Among them, there is Sanger, Roche 454, Illumina, Ion Torrent, PacBio and Oxford Nanopore, which will be described below. All these platforms achieve the same objective but with a different approach to detecting the sequence of genetic material. Among them are 1st (Sanger), 2nd (Roche 454, Illumina and Ion Torrent) and 3rd (Pacific Bioscience (PacBio) and Oxford Nanopore) generation sequencers.

B. NGS

The second generation of sequencers all followed the same principle as the Sanger method, consisting of incorporating a specific dNTP one after the other. Still, in a second-generation sequencer, it does not stop the elongation process. Unlike the Sanger method, the second generation of sequencers starts by fragmenting the DNA into small fragments (called "reads"). A few years after the Roche 454 was introduced, a new generation of sequencers (the thirdgeneration) was created. The main advantages of the third-generation sequencer are that it produces longer reads and does not require a DNA or RNA amplification step. Longer reads have the advantage of being able to contain an entire region of the genome in a single fragment.

In contrast, small ones must be assembled to reconstruct the same region. Third-generation sequencers sequence each fragment generated from DNA or RNA molecules from a sample in real time, as there is no amplification step.

PacBio Single-Molecule Real-Time (SMRT) sequencing can generate fragments above 10 kilobases (kb) and uses the detection of nucleotides by the emission of waves specific to each nucleotide (Figure .22.A.). The Oxford Nanopore is a technology consisting of a protein with a nanopore embedded in an electrically resistant membrane and coupled to a sensor chip. This nanopore is immersed in an electrolyte solution, which causes an ionic current to be generated.

The sensor chip underneath the nanopore can detect any changes in the ionic current that may occur. Thus when DNA pass through a nanopore, a specific electronic signal is emitted and detected (Figure. A. Different steps in the Roche 454 sequencing technique. First, the DNA or RNA strand is fragmented, then the primers are bound to both sides of the reads and each primer is attached to its specific beads. The next step is emulsion pcr where each read on the bead is amplified and attached to the bead in the same way. The beads are then placed on a picotitration plate so that sequencing can begin, a light is emitted when a specific dNTP is bound to the complementary strand, allowing identification of the nucleotides and the order in which they occur in each read [START_REF] Siqueira | Pyrosequencing as a tool for better understanding of human microbiomes[END_REF]. B. Description of the Illumina sequencing process with the fragmentation of the DNA or RNA strand into 150-300 bp reads, hybridisation with primers on either side of the reads and then onto complementary primers on the flow cell. Then the Production of a complementary read that binds directly to the primer on the flow cell is performed followed by a first clean-up step that removes the first reads that have bound to the flow cell. A bridge is formed by the second primers attached to the reads that have bound to their complementary primer on the flow cell, followed by bridge amplification forming clusters for each read. A further clean-up step is performed to cleave one of the read strands, the next step is the start of sequencing, the polymerase allows specific dNTPs coupled to a fluorochrome to be attached to the primer and a laser is turned on to detect the fluorochrome whose emitted wavelength allows identification of the nucleotide to which it is attached, thus the sequence of the reads is determined [START_REF] Chaitankar | Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research[END_REF]. C. How the Ion Torrent sequencer works. The first step is to create a DNA or RNA fragment and to ligate the primers on both sides of each fragment. One of the two primers for each read will be attached to a bead and a PCR will be performed to amplify the number of identical reads on each bead. Then each bead will be placed in a well of the micromachine with one bead per well. A solution containing a polymerase and a specific dNTP is incorporated into each well, causing the initiation of complementary strand production. When the specific dNTP in the solution is bound to the DNA or RNA template, the reaction produces the formation of H+, resulting in a pH change in the solution.

Each well is connected to a pH meter and so when the pH changes, the pH meter detects this and records the information of the pH change when a specific dNTP is added to the solution. A clean-up process is carried out between each incorporation of a new specific dNTP to remove the excess of previous dNTPs. When a single dNTP is added, it produces a limited amount of H+ and the more dNTPs that are added, the more H+ are produced and thus detected. Thus, if the reading contains a repeat of the same nucleotide several times in a row, it will be detected by the pH meter [START_REF] Golan | Using state machines to model the Ion Torrent sequencing process and to improve read error rates[END_REF].

(99,100) A. How PacBio technology works, with a first step of fragmenting the DNA or RNA and then ligating these fragments to primers on both sides. Then, each fragment is incorporated into a SMRT consisting of millions of ZMWs, each of which will host a fragment. The sequencing process can then begin by incorporating a polymerase and a specific dNTP coupled to a fluorochrome into each ZMW. The attachment of the fluorochrome to the complementary label results in the emission of light which is detected and recorded [START_REF] Metzker | Sequencing technologies -the next generation[END_REF]. B. Operation of the Oxford Nanopore sequencer. A nanopore protein coupled to a helicase is embedded in an electrically resistant membrane which is connected to a detector chip just below it. The DNA or RNA is fragmented into long reads and ligated with adapters on both sides, and these reads find their specific adapters in the nanopore. Then the single strands of DNA or RNA pass through the nanopore, each nucleotide passing through inducing a compositionspecific change in the ionic current that is detected by the sensor chip (100).

NGS conducted new bioinformatic challenges from the storage needed to stock data and compute them to analysis of data issues from sequencing. First, a raw data analysis is made to control the quality of the sequence. Outputs of the sequencer are read in a FASTQ file that gives the nucleotide composition for each read and a score of quality for each base contained in a sequence based on the Phred score (Q), which is indicated below each base by an ASCII character and is comprised between 4 and 60 with a higher score representing greater assurance on nucleotides concerned. The Phred score is calculated from the measurement of the strength of the signal for each base compared to the noise, and it gives the probability of error identification (e) according to this formula:

𝑃 (𝑒) = 10 𝑥 -𝑄/10
The Phred score is used to eliminate poor-quality reads or portions of reads, also called trimming. Another raw analysis step concerned other elements, such as Guanine and Cytosine content percentage. Furthermore, to ensure the quality of the sample, it is possible to look into the depth of sequencing and coverage. Depth represents the total number of reads sequenced for a sample, while coverage is the total number of nucleotides sequenced divided by the total number of molecules detected in a sample. Thus, better coverage allows for more confident identification of a DNA or RNA sequence, sequencing depth increases the chance of sequencing all molecules in a given sample and improves confidence in each identified nucleotide and its positions and in microbial diversity estimation. All sequencing techniques allow very qualitative identification of sequences with strengths and limitations (Table.4). For example, concerning depth and coverage, generally, the second generation of sequencers allows for higher sequencing depth because billions of short sequences could be sequenced in a single run. However, because the third-generation sequencers generate longer reads to give greater confidence in the identification and position of individual nucleotides, it is potentially unnecessary to have as great a sequencing depth as that found in the second-generation sequencers.

On the other hand, third-generation sequencers are better able to retrieve regions that are usually difficult to analyse, such as highly repetitive regions, as the read length could extend beyond the repeated region (Table .4). In conclusion, the preference for one technique over another depends on the question being asked. While 16SrRNA analysis identifies higher taxa levels than species, shotgun analysis allows identifications at the species and even strain level. Therefore, although the 16SrRNA study provides a good picture of the microbiota composition, the shotgun analysis provides a more accurate one. However, it is essential to consider that with 16S rRNA sequencing, there is little risk of sequencing host DNA, or at least much less than the shotgun, as the target region is only present in prokaryotes. On the other hand, shotgun sequencing has a higher risk of host contamination, especially depending on the sample type (faeces from healthy donors are not significantly contaminated by host DNA, unlike saliva). If the host DNA dominates the microbiota DNA, shotgun sequencing will only offer a poor representation of the microbial species in the sample concerned. It is essential to consider this point when choosing the method depending on the material on which the work is done.

c. Control quality of the sample

Furthermore, when sequencing DNA or RNA, there are two ways of treating sequence data to report them to a specific microorganism: doing de novo assembling or mapping sequence into a reference database. Many reference databases exist for 16 rRNA sequences, so de novo assembly is mainly used when the sequences come from shotgun sequencing. De novo assembly requires powerful computer tools, people who know how to use them, and it takes time. Thus, the risk is that if shotgun sequencing data does not have an optimal match with reference databases and there is no possibility of de novo assembly, the representativeness of species within the microbiota will be low.

d. 16SrRNA VS shotgun i. Microbiota representativity

The advantage of the shotgun focuses mainly on the fact that it is possible to theorise about the functionality of the microorganisms present in each sample which is a powerful asset. It gives the diversity of the species in a sample and information on what they might produce (as a metabolite, for example) and thus contribute to the ecosystem. Tools for predicting functions based on the taxa present in a sample were initially only available for shotgun samples. Still, programs have recently been developed to apply this method to 16S rRNA sequences but are mostly limited to inferring a species' core functions (genes shared by a vast community of species). However, shotgun metagenomics remains the most accurate way to predict functional genes against 16S rRNA sequences as it could explore both the core and all accessory (mobile element of the genome) functions that are strains specific (101). In studying the ecosystem as a whole, the shotgun provides a significant strength in understanding interactions.

However, the cost of the two analyses is not the same, 16SrRNA is much cheaper than the shotgun, making it the most widely used technique in microbiota studies (at least 5 to 10 times cheaper based on 2023 prices). Furthermore, the advantage of 16S over a shotgun is that the data analysis is simpler. Sometimes the large amount of information provided by the shotgun can be difficult to sort out and try to extract information that may be relevant from that which may be less relevant. It is important to have a good idea of the information each technique can provide and their relevance to the research project to make a choice.

As with any experiment in biology, the only good technique is the one that answers the question asked in the first place. Knowing all the possibilities to make the most appropriate choice is important.

Sequencing has improved our knowledge of ecology. In particular, it has made it possible to calculate the diversity of microbial communities more precisely. The concept of an a b g diversity index to reflect the richness of the different microbial species present in a specific sequencing, all these measures were based on observation, which was limited; sequencing has made it possible to reflect diversity better.

In the medical field today, these different indexes are frequently used; sequencing with the techniques that followed Sanger sequencing has been a quantum leap in advancing microbiota studies in human health, among others. It made it possible to know the richness and abundance of each microbiota identified in humans, compare them, and know what they can contribute.

The human organism has much microbiota that lives on the surface of different organs. Each microbiota carries the signature of the environment in which it evolves, so they are all distinct

Microbiota in humans

A. Focus on the gut microbiome.

in composition. The digestive system contains the greatest diversity in terms of microbial composition, with microbial biomass estimated at 10 11 microbial cells per mL. Throughout this organ set the physiological conditions change with oxygen distribution becoming poorer as one moves down the anal canal. The colon, the organ where oxygen is most scarce, has the largest microbiota in quantity in the entire human body. Under these conditions, the species most likely to grow are strict anaerobic species (which grow in the absence of oxygen) and facultative aerobic-anaerobic species (which can grow under anaerobic conditions although they prefer aerobic conditions). The high diversity of microorganisms in the colon can potentially be explained by the fact that the nutrient source for the microorganisms (faecal matter in the colon), remains longer in this organ than in other compartments of the digestive tract. This is where the fermentation of the remaining food takes place, particularly by bacteria. Thus, many of the species identified present in the colon are determined as crucial to maintaining the health of the organism.

(102) This phylogenic tree comes from dataset comprising 13,133 human gut microbiome analyses from 75 different studies. This tree shows many species, most of which are unidentified metagenomic species (UMGS) compared to those found in the human gut reference (HGR). The outer layer (green bar) represents the genome prevalence in the 13,133 datasets. The gut microbiota shows great diversity within and between individuals. It is therefore important to be representative of as many people as possible, and currently people from Africa and South America are under-represented in microbiota studies. This tree also highlights the fact that it is difficult to interpret metagenomic studies when many species are still unknown. It should also be considered that the majority of microbiota studies focus on bacteria, and moreover on the most abundant species, which can create biases in the interpretation (102)

These different microorganisms are so numerous that there are still many that remain unidentified today at the species scale which is one of today's great challenges (Figure .23). The identification of microorganisms and their classification is a very rigorous and important discipline based on the similarities that the newly identified species share with others already known. Through these attributions, called taxonomy, a genetic sequence is assigned to a particular species or higher taxa during sequencing. Thus, each living being, including microorganisms, is classified according to taxonomy, which distinguishes 9 taxa from the broadest to the most precise: the domain, the kingdom, the phylum, the class, the order, the family, the genus, the species and finally the strain (Table.5).

As mentioned above microbiota is a consortium of microorganism including bacteria, fungi, virus protist and archaea, they live together, but their repartition is unequal, some has been identified as the dominant population of microorganism in human microbiotas. Indeed, bacteria seem dominant compared to fungi, viruses, archaea and protists. The high proportion of bacteria explains why gut microbiome studies focus mainly on this population of microorganisms. Information on other microbial populations is beginning to attract the interest of researchers who have started exploring other communities' composition in recent years.

There are two main phyla of bacteria, the Bacteroidota and the Firmicutes, the best known and most studied. Although studies on the gut microbiota are recent and much remains to be understood about its dynamism and relationship with the host, it is now clear that its presence is inextricably linked to a state of homeostasis in the digestive system. So, although the microorganisms residing in the intestinal tract are referred to as commensals, the most appropriate term to refer to them should be mutualistic symbiosis. This leads to the concept that humans form a holobiont with their microbiota which is expressed in the different stages of an individual's life. While the host is known to be a habitat and a place rich in nutrients for the microbiota, what the microbiota brings to the host has been less clear until now. It is, therefore, easier to understand how the host can shape the gut microbiome (depending on physiological state and nutrient intake), than the other way around. Advances in research have led to a better characterisation of its importance and impact.

The digestive system forms between the third and 32nd week of pregnancy (103). During this time, immune mediators and metabolites (including microbial metabolites) are transmitted from the mother to the foetus via the placenta. This transmission is involved in the development of the immune system and possibly also in the formation of the foetal digestive tract. There are still questions about the timing of the colonisation of microorganisms. Some studies have developed the idea of a sterile uterus, while others have shown the presence of microorganisms in the meconium of the foetus (104,105). Research should continue to develop to understand better the timing of first colonisation and when the influence of microorganisms on the development of the gastrointestinal tract and the immune system begins.

The implantation of microorganisms is a complex dynamic that is not fully established today.

It seems that the stronger the interaction between microorganisms, the lower the microbiota diversity. Thus, there is a greater chance of diverse microbiota when many species that do not share a strong bond colonise the environment simultaneously rather than just a few species that share very strong bonds (106). Some animals pass on their faecal microbiota to their offspring, which facilitates the establishment of a diverse microbiota. In humans, the major colonisation occurs just after birth with bacteria from the mother's vaginal and faecal microbiota and the

B. Gut ecosystem a. Gut ecosystem formation

i. Digestive tract environment (for babies born vaginally) or the skin microbiota and the environment (for babies born by caesarean section). The baby's birth, therefore, determines which species will first colonise the newborn's digestive tract. After birth, the transmission of glycans through the milk and their production in the epithelium also strongly influence the species that can colonise the digestive tract of the newborn (106). This implantation, which takes place during the first weeks of life, is crucial because the newborn's digestive tract is structurally and functionally still immature and its evolution seems to depend on many factors, including the gut microbiota.

Indeed mucosa and intestinal mucous haven't finished their development and seem to need contact with gut microbes to have an optimal one (107).

The first immune system developed by vertebrates was the innate immune system, followed by the adaptive immune system. The development of the adaptive immune system was long considered a reinforcement of the innate defence system, but a new hypothesis has recently emerged (108). According to this new hypothesis, the colonisation of microorganisms in the host plays a role in the development of an adaptive immune system. These latter would evolve jointly, with a specific response to each species colonising the host and a recognition of which species may be favourable and which may be pathogenic, and therefore which should be eliminated. By selecting microorganisms that should be eliminated from those that should not be eliminated, the adaptive immune system must prevent the innate immune system from initiating elimination processes towards all microorganisms. This phenomenon implies the establishment of communication between the two immune responses. The initiation of this theory came from observing the diversity of the microbiota between invertebrates, vertebrates, and vertebrate gnathostomes. The population with the most diverse microbiota is the vertebrate gnathostomes, the only ones with an adaptive immune system, suggesting that only animals with a specific immune response could have high diversity in their microbiota. Furthermore, the development of the adaptive immune system parallels the colonisation of the microbiota in the gut, reinforcing the link between the adaptive immune system and the microbiota. This hypothesis is supported by the fact that just after birth, i.e. at the time of the major colonisation of the host by microorganisms, the epithelial cells of the intestine, but also the immune cells, acquire receptors called pattern recognition receptors (PRRs) which can be membrane-bound, in this case, the Toll-Like Receptor (TLR) or the C-type lectin receptor (CLR), or cytosolic, such as the NOD-like receptor (NLR). All these receptors have the particularity of recognising ii. Immune system molecular motifs present only on microorganisms and called microbe-associated molecular motifs (MAMPs) (108). The recognition of MAMPs by NLRs from the earliest stages of life contributes to the maturation of the second lymphoid tissues that play a crucial role in the mutualistic relationship between the host and the microbiota. This function is partly explained by the fact that the second lymphoid tissues are at the interface between the microbiota and the host tissues, which explains why an immune response can be rapidly set up in the event that a pathogenic microorganism attempts to cross the mucosal layer to the epithelium. The role played by the microbiota in the development of these structures was confirmed in an experimental study of germ-free microorganisms, whose secondary lymphoid tissue was less developed than that of the control group. This difference appears to be attenuated when germfree mice are colonised by Bacteroides fragilis, which produce polysaccharides A (PSA), resulting in the maturation of their secondary lymphoid organs, which resemble wild-type animals (109).

The intestinal microbiome can induce mechanisms essential for the intestinal ecosystem's proper functioning. They occur through contact between the components of the microorganisms and the products they synthesise. Some of these mechanisms have been identified and described.

As mentioned above, the gut microbiota is involved in the maturation of the intestinal mucosa, which is a major component of the intestinal barrier protecting tissues from elements passing through the intestinal lumen. The disruption of this barrier could cause an imbalance in the homeostasis of the intestinal ecosystem, as it is one of the keystones. Various mechanisms, which can be direct or indirect have been highlighted concerning the role of the microbiota in maintaining this intestinal barrier.

b. The gut ecosystem in homeostasis i. Relationship between microbiota and tissue cells

The intestinal microbiota acquires great stability thanks to its mutualistic symbiosis within the host. This stability contributes to the barrier. The intestinal lumen's ecological niche has limits and cannot accommodate all the microorganisms that could pass along the digestive tract. In the same way, it would be difficult for a species that is part of the microbiota, which is called commensal, to see its population grow because it would be at the expense of another population. However, there is no reason why this should happen in a stable microbiota as long as there is no change in the ecosystem that would select the growth or establishment of one species over another. In this way, under conditions of homeostasis, developing a species that could prove pathogenic for the host can be prevented by this competitive effect.

Each microorganism species can produce many metabolites, some of which have antimicrobial properties. These metabolites regulate the microbial populations of the gut microbiota and prevent the colonisation of the gut microbiota by exogenous species that may be pathogenic.

Among them is a peptide such as Ruminococcin C1 produced by the species Ruminoccocus gnavus, which is thought to have broad-spectrum antimicrobial properties and little impact on gut tissue (110). Secondary acids bile, a product of deconjugating primary acids, may also have antimicrobial effects. Bile acids synthesised from cholesterol in the liver cells are called primary acids and are mainly represented by chenodeoxycholic acid (CDCA) and cholic acid (CA). These two primary acids are coupled to glycine or taurine and, once produced, are stored in the gallbladder between meals. When the duodenum receives chyme (food partially digested by saliva and the stomach), the gallbladder gets a message that releases bile (formed from primary acids) into the intestine. Bile plays a role in the absorption of fats and vitamins. Once (TNFa) and Interferon gamma (IFNg) together with FICZ for 48 hours prevented the morphological disruption of the TJ compared to cells that were not exposed to FICZ (112). The microbiota does not exclusively produce some of the metabolites involved in the epithelial barrier; some could also be the product of host synthesis, such as polyamines.

The host can produce polyamines in their cytoplasm through enzymes such as ornithine decarboxylase and transferase in the upper gut. In contrast, the gut microbiota degrades polyamine-rich foods into polyamines in the lower gut. In an in vitro experiment, host polyamines correlated with increased paracellular permeability. To establish this mechanism, researchers exposed intestinal epithelial cells (IECs) to a blocker of polyamine biosynthesis (a-difluoromethylornithine) which resulted in a decrease in free Ca2+ in the cell cytoplasm and, consequently, a reduction in the expression of E-cadherin, a protein involved in intercellular adhesion, and thus an increase in paracellular permeability (113). The authors published another article in which they added spermidine to the same model (where IEC-6 was exposed to a-difluoromethylornithine). Adding this polyamine led to an increase in Ca2+

within the cell and in E-cadherin; they also showed a decrease in the paracellular permeability (114). The mechanisms established in gut cells involving the synthesis of host polyamines could perhaps be applied to polyamines produced by the microbiota in the large intestine.

The mucus is one of the components of the epithelial barrier and is composed of proteins called mucins that are synthesised by the goblet cells in the intestinal epithelium. The mucus layer differs along the digestive tract, as does its composition. It has also been shown that the proportion of goblet cells to the total number of epithelial cells is greater in the colon than in the small intestine). Correlations have thus been established between the number of microorganisms living in the lumen and a higher proportion of goblet cells. It appears that the greater the presence of microorganisms, the higher the proportion of goblet cells in the epithelium, which could lead to the hypothesis that the microbiota may have a role in mucin production by goblet cells. Some studies show that components of the microbiota could contribute to this production. One study uses germ-free mice colonised with the caecal contents of wild-type mice. The germ-free mice have a thinner and more porous mucus layer. The

(iii). Induction of mucus production in epithelium

colonisation of the caecal microbiota of mice with normal mucus composition allows germfree mice to normalise their mucus in the colon within six weeks of colonisation. This indicates that the microbiota may contribute to developing and maintaining mucus layers in the colon (115).

The involvement of microbiota in epithelial cell stimulation was raised in a 1993 study which showed that in germ-free animals, there was a mitotic index and a decrease in cell turnover compared to the wild-type (116). In 2021 a study investigated the mechanisms underlying the induction of cell proliferation in colonic stem cells (CSCs) and whether the microbiota could be a player in these mechanisms. Using a spheroid model, they demonstrated that NOX1 was directly involved in proliferation by activating the EGFR pathway. They tried to see how NOX1 expression was regulated in CSCs. Using an ex vivo model of the proximal or distal spheroid, they showed no difference in the maintenance of NOX1 expression between the two and concluded that there was a gap. As in vivo NOX1 expression is positively correlated with the gut microbiota along the intestinal tract, this implies that the gut microbiota could be the reason why they could not see a difference in NOX1 expression in the proximal or distal colon spheroids. In their culture, they added microbiota-derived TLR ligands such as liposaccharide (LPS) and showed that spheroids from wild-type animals grew more than spheroids from NOX1-/-animals. To confirm the involvement of the microbiota in NOX1 production by CSCs, they administered different types of antibiotics to the animals and measured NOX1 expression in the colon. They found that NOX1 expression was lower when fewer bacteria were present in the distal colon only. They then examined the proliferative capacity of the cells by flow cytometry in these same mice. While there was a difference between wild-type and NOX1-/mice about cell proliferation, no difference was found after these two groups of animals were treated with a wide range of antibiotics.

Furthermore, when the microbiota of conventionally bred mice was implanted into germ-free co-infected mice, a proliferative response was recorded after one week of implantation. It lasted for up to 4 weeks after implantation (117). This study highlights the importance of the presence of microorganisms in the proliferation of colonic stem cells and, thus that the microbiota under homeostatic conditions participates in the cell renewal of the epithelium.

(iv). Colonic epithelium cellular renewal

Epithelial cells can produce metabolites with antimicrobial properties called antimicrobial peptides (AMPs). The production of AMPs by epithelial cells can occur in several ways, one of which is the stimulation induced by the contact of their TLR with certain microbiota species.

Some AMPs, such as C-type lectin, defensin and cathelicidin, have antimicrobial properties that act directly on microorganisms. Others act indirectly by depriving the microorganisms of elements essential for their development (lipocalin2 and iron, an element necessary for the growth of certain bacteria).

In conclusion, the gut microbiota plays a multiple roles in the homeostasis of the gut ecosystem (Figure .24). Their communication with the intestinal tissue contributes to maintaining a healthy state in the gut. Still, it is not only the host mucosal cells that communicate with the microbiota. Immune cells also play an essential role in the host-microbiota mutualistic symbiosis.

(v). Stimulation of epithelial cells' antimicrobial properties

Figure 24. Role gut microbiota in the formation and/or maintain of the epithelial barrier.

Influence comes directly from microbiota with his only presence via competition and his production of metabolites. Action can be due to his influence on gut cells on with the induces production of mucus, cell renewal and production of antimicrobial peptide.

As with its relationship with the host, the gut microbiota can regulate the immune system through the elements it produces and its components.

Microbial metabolites can be produced in three ways: degradation of molecules from nutrition, degradation of components produced by the host and metabolites produced directly by the microorganisms.

Among the microbial metabolites, the best known are short-chain fatty acids (SCFAs). SCFAs are produced by microorganisms via the fermentation of fibre from the diet. SCFAs appear to influence the immune system through different mechanisms. They may act on IL-10-producing cells. IL-10 is a crucial anti-inflammatory cytokine that is important in various studies. When humans or animals are deficient in IL-10 or its receptor, this appears to lead to severe intestinal inflammation (118,119). SCFAs affect the LTCD4 cell population, their regulation, and their cytokine production. This population of lymphoid cells may be IL-10-producing and, therefore, crucial for regulating the immune system response. For example, in a study on Specific Free Pathogen (SPF) and Germ-Free (GF) animals, it was shown that consumption of saturated fatty acids alone (propionate, acetate or butyrate) or in combination increased the number of LTregs expressing FOXP3 and IL-10 by involving the GRP43 or FFAR2 receptor (120). Another study looked at the effect of SCFAs on the GRP43 receptor. They showed that the interaction between SCFA and GRP43 in TH1 cells could decrease excessive inflammation through increased IL-10+ T cell production (121). Another cytokine identified as essential for preventing inflammation in the gut is IL-22. One study investigated the relationship between saturated fatty acids and IL-22 production by LCD4 cells and intestinal ILCs. They demonstrated that the binding of butyrate to GPR41 on LTCD4 cells induced IL-22 production. They also showed that butyrate-induced IL-22 production by LCD4 cells and intestinal ILCs is crucial for tissue protection against C.rodentium infection. Finally, the authors verified the results obtained in mice on patients and established that in IBD patients, butyrate intake increases IL22-producing LTCD4 cells and IL-22 concentration (122). In addition to their effects on LTCD4 cell

ii. Relationship between microbiota and immune system

(i). Microbial metabolites

From nutrition populations, SCFAs may also act on LTCD8s as they form memory LTCD8s, thus allowing a more rapid immune response to an infection by an antigen already encountered by the immune system (123). SCFAs are a large family of molecules, including lactate, that bind to the GRP31 receptor of CX3CR1+ phagocytic cells and trigger the protrusion of the dendrites of these same cells, which can then capture microorganisms present in the intestinal lumen without having to leave the tissue (124).

Secondary bile acids produced by microorganisms affect epithelial cells, as shown below, but some researchers also highlighted the impact it could have on immune cells. Some of them have studied the action of taurocholic acid (TLC). To do this, they exposed primary human macrophages to LPS, as well as to LPS and taurocholic acid. They found that the production of IL-6, TNF-a, IFN-b and IL-12 increased in macrophages exposed to LPS alone, while it did not increase in macrophages exposed to LPS and taurocholic acid. This study indicates that the phenotype of macrophages induced by TLC exposure is one where the IL-10/IL-12 ratio increases, where IL-10 production remains identical to that of macrophages not exposed to TLC (only to LPS), but where IL-12 production is somewhat decreased. In addition, this macrophage phenotype is reported to have reduced phagocytic activity and inflammatory cytokine production so that its profile resembles that of an M2 macrophage more than an M1 macrophage. Hence, secondary bile acid as TLC seems to avoid inflammatory response in macrophages when exposed to a constituent of microorganisms which could lead to tissue degradation (125).

Microorganisms produce de novo various metabolites that could be useful to the host. Some of these directly impact the immune response, such as branched-chain amino acids (BCAAs), did not affect the ability to differentiate into Tregs. The authors then compared the turnover capacity of splenic CD4+ FOXP3+ T cells on a normal or isoleucine-deficient diet with bromodeoxyuridine (BrdU). They found that BrdU was lower in splenic CD4+ FOXP3+ cells for animals fed the isoleucine-deficient diet compared to the control. The authors also measured the activation of the mTOR pathway in the same group of animals and showed that it was reduced in animals fed a low isoleucine diet. To understand whether this mechanism could affect susceptibility to inflammation, they treated both groups of animals with DSS treatment.

They found that the group fed a low-isoleucine diet developed more severe inflammation than the control group. This paper concluded that isoleucine helps to increase the turnover capacity of Tregs, which is essential to prevent the development of severe intestinal inflammation, via the mTOR pathway (126).

Another example could be polyamine spermidine. Spermidine is one of the essential polyamines microbiota produced together with putrescine. In the upper intestine, the presence of this polyamine is mainly due to the diet, but in the colon, it is attributed to the microbiota.

Both polyamines are precursors of other polyamines, such as spermine. In an in vitro study, Jeong et al., exposed murine macrophages to lipopolysaccharides (LPS) that induce the production of nitric oxide (NO) and prostaglandin E2 (PGE2), which are mediators of inflammation and added spermidine to the culture. The addition of spermidine decreased the production of NO and PGE2 by the macrophages. They also studied the effect of spermidine on the LPS-induced production of inflammatory cytokines: TNFa and IL1b. While murine macrophages exposed to LPS increased their production of TNFa and IL1b, the addition of spermidine attenuated this increase. The same experiment was replicated concerning reactive oxygen species (ROS), and the same dynamics occurred, meaning that when murine macrophages were exposed to LPS and spermidine, the level of ROS they produced decreased compared to murine macrophages exposed to LPS alone. The downregulation of ROS and NO was confirmed in vivo in a zebrafish model that received LPS and spermidine. Finally, after exposure of zebrafish larvae to LPS, the authors showed that spermidine decreased the accumulation of neutrophils and macrophages. This study indicates that spermidine could have a modulating effect on the immune system and, in particular, on innate immune cells (127).

It is not only microbial products that influence the immune response; the mere presence of constituents of microorganisms also regulates the immune response.

When a ligand activates a TLR, downstream adaptor molecules are recruited, for example, myeloid differentiation factor 88 (MyD88). This results in a call to other downstream molecules, leading to a cascade reaction that results in the transcription of genes involved in the immune response, such as cytokines, chemokines or costimulatory molecules. (128). Some of the microbiota constituents can bind to specific TLRs and cause a cascade reaction involving MyD88 that ultimately leads to a decrease in the passage of phagocytes from the lamina propria to the mesenteric lymph node. The binding of a microbial constituent can also lead to the production of an anti-inflammatory cytokine, for example, when the bacterium Clostridium butyrum (CB) binds to the TLR2 of CD11+/CD11c/F4/80 macrophages, leading to the production of IL-10 involving the MyD88 protein. In a DSS-treated SPF mouse model, the administration of CB alleviated the severity of symptoms caused by DSS. The mechanism of action of this bacterial strain was demonstrated using different animal models, including RAG2 -/-and IL10 venus (to study the expression of the IL10), which confirmed that the attenuation of colitis by CB did not involve lymphoid cells but CD11+CD11cF4/80 macrophages where the most significant IL-10 gene expression was identified. To determine which pathway is involved in IL-10 production, the authors isolated mononuclear cells from the lamina propria of TLR2-/-and MyD88-/-mice that were treated with DSS and added heatkilled CB to the culture and compared IL-10 production to that of the wild type and found that IL-10 production was greater for the wild type mice. Finally, they used strains of mice whose macrophages could not produce IL-10 and showed that, for these mice, CB treatment did not decrease the severity of the pathology induced by DSS treatment (129).

One study was interested in understanding the regulatory mechanism of phagocyte turnover that allows myeloid cell homeostasis. Hergott et al. hypothesised that the gut microbiota might be involved in this unknown mechanism. To do this, they measured the turnover of neutrophils and inflammatory monocytes in the blood of mice after treatment with a broad-spectrum antibiotic or gut-restricted neomycin, which appears to act on bacteria that have a component that is a ligand for the NOD1 receptor. Next, mice were injected with BrdU and examined BrdU+ neutrophils and inflammatory monocytes on different days after treatment. In both antibiotic-treated groups, there were fewer BrdU+ neutrophils and inflammatory monocytes than in the wild-type group, meaning that neutrophil and inflammatory monocyte turnover is lower in the antibiotic-treated group. In addition, both antibiotic treatments decreased the number of neutrophils and anti-inflammatory monocytes in the blood, spleen and bone marrow.

(ii). Microbial constituents

This experiment concludes that antibiotics reduce the life span of these cells. A metagenomic study on neomycin-treated mice showed that the Lactobacillae family decreased, and this family is known to have peptidoglycans that bind to NOD1, so the question was whether NOD1 could be linked to the lifespan of specific myeloid cells. The authors studied the life cycle of phagocytes ex vivo by measuring the percentage viability using annexin V in wild-type, Nod1 - /-, Nod2Tlr2 -/-and Tlr4 -/-mice. While phagocytes from wild-type, Nod2Tlr2 -/-and Tlr4 -/-mice had the same percentage of phagocytic viability, Nod1 -/-showed an increase in the proportion of phagocytic cells undergoing apoptosis. To test whether NOD1 still has this effect in vivo, they injected NOD1 ligand into neomycin-treated mice, which resulted in phagocytic viability similar to that of the control. Since the absence of a particular type of microbiota component leads to increased apoptosis and clearance of neutrophils, it seems that this component is essential for regulating the life span of these cells under homeostatic conditions (130).

The mechanisms described below are just a few examples of the shared relationship between the gut microbiome and the host. Although the mechanisms described so far do not capture the entire relationship between the two entities, they help understand how inseparable they are. As mentioned earlier, in a healthy state, the host and the microbiota share a mutualistic relationship within the gut ecosystem. It is, therefore, understandable to hypothesise that if something goes wrong with one of them, it could influence the other. For example, a change in the host's physiology or the microbiota's ecology could disrupt the gut ecosystem and, thus, the development of pathology. Dysbiosis is a breakdown in the mutualistic relationship between the host and the gut microbiota. It is defined as a disturbance in host parameters (inflammation, permeability, oxidative stress) and the microbiota's composition. Depending on the extent of the disorder (on both sides), the return to a healthy balance is more or less complicated. In the context of pelvic radiotherapy, the effects of ionising radiation on the healthy cells of the colon have been discussed previously, but it seems that a change in the microbiota has also occurred.

In Thus, the release of ROS into the gut ecosystem after exposure to ionising radiation led to a host cell response. Due to the proximity of the gut microbiota, exposure to ROS seems inevitable. In the context of the link between gut microbiota composition changes and mitochondrial genome changes, a study has attempted to explain this correlation better. To do this, they used a strain of mice whose mitochondrial genome had been mutated, increasing ROS production. They then compared the microbiota diversity between the different strains of mice using the Shannon index and the UniFrac weighted distance. The mice showed differences in microbiota diversity depending on their mutation. They also compared the production of ROS between a mouse strain with high a-diversity and a mouse strain with low a-diversity. They validated their hypothesis that the higher the a-diversity, the lower the ROS production. They did not look for a mechanism. However, they hypothesised that the change in microbiota could be due to dysmotility. This dysmotility would be due to a modification of 4. Pathology in the gut ecosystem: the influence of gut microbiota A. Changing conditions in gut ecosystem mitochondrial metabolites or to dysfunction of redox signalling in the mitochondria leading to a defect in the function of neuronal and smooth muscle cells. The last hypothesis is the impact of mitochondrial dysfunction that led to the alteration of the balance of Treg and effector T cells that maintain the inflammatory process and thus remodel the gut microbiota (134).

Because, as mentioned above, non-cancerous tissue exposure to ionising radiation led to the production of ROS, and from this study presence of ROS could impact gut microbiota composition, it would be interesting to see if it is impaired after pelvic radiotherapy.

There are a few clinical studies concerning the dynamics of the gut microbiota before and after pelvic radiotherapy in cancer patients. In these different studies, finding a specific signature of a microbiota living in an ecosystem exposed to ionising radiation is challenging. It can be explained by the diversity of the patient profiles selected in these different studies. Indeed, the patients do not have the same type of tumour, do not receive the same kind of radiotherapy (often not alone and with drugs) nor the same dose, are not of the same sex and do not have the same diet. Two different trends emerge when considering the clinical studies where patients only receive radiotherapy treatment during the study period. Only three studies examined an index reflecting the diversity within samples. Two of the three studies created groups within groups of patients who received radiotherapy based on their symptoms. The diversity in these studies appears to change, but not in the same way. One study focused on men who received pelvic radiotherapy for high-risk prostate cancer. This study examined the Chao index before, during and after 12 weeks of radiotherapy according to the severity of the patient's symptoms.

Thus, patients were divided into three groups: the first without symptoms, the second with nonpersistent symptoms and the last with persistent symptoms. The severity of symptoms was assessed twice, once by the patient and once by the clinician. However, four out of five times, analysis information was missing for the assessment of severity, both for the patient and the clinician. Specific diversity by symptom severity only differed when patients reported their symptoms. Indeed, patients without symptoms showed a decrease in the Chao index over time, and patients with persistent symptoms showed an increase in the Chao index over time. In terms of taxon evolution, patients with persistent symptoms showed a decrease in Clostridium B. Composition of gut microbiota after pelvic radiotherapy a. Clinical studies IV and Roseburia (135). The second study involved 11 patients with cervical, anal or colorectal cancer. They separated the patients into two distinct groups: those who developed diarrhoea (n=6, with four women and two men) either during radiotherapy or after the end of treatment and those who did not (n=5, all women). They worked on these two cohorts and examined the a-diversity with Chao and Shannon index. They showed that before radiotherapy, there was no difference between the three groups in the Chao. For the Shannon index, the patients who developed diarrhoea already had a lower Shannon index than the other two groups. After radiotherapy, the Chao index was decreased only in patients who developed diarrhoea. The Shannon index decreased in both groups of patients after radiotherapy, more significantly in the diarrhoea group than in the non-diarrhoea group. They also compared the ratio of the two most representative phyla of the human gut microbiota, the Bacteroidota (Bacteroidetes) and the Firmicutes. They showed that the ratio of Firmicutes/Bacteroidota was higher in both groups than in healthy patients (with an increase in Firmicutes and a decrease in Bacteroidota). After radiotherapy, this ratio seems to decrease, but there is no clear statistical difference. They compared the abundance of specific genera before and after radiotherapy between the three groups. Before radiotherapy, the genus Bacteroides was lower in patients receiving radiotherapy than in healthy patients. After radiotherapy, Bacteroides increased in patients who received radiotherapy and more significantly in patients who developed diarrhoea.

For the genus Faecalibacterium, the trend is the opposite. Before radiotherapy, this genus was present in more significant quantities in patients who received radiotherapy than in healthy subjects. After radiotherapy, the presence of Faecalibacterium decreased in patients who received radiotherapy. Finally, for Lachnospiraceae, only a comparison before and after radiotherapy was performed in patients who received radiotherapy. Compared to before radiotherapy, the genus Lachnospiraceae decreased in both groups of patients who received radiotherapy and, to a greater extent, in patients who did not develop diarrhoea. In addition to the microbiota, the authors measured the fatigue that patients experienced during their radiotherapy treatment. The onset of fatigue occurred at the same time as diarrhoea for the group of patients concerned, and their fatigue appeared to be greater than that experienced by patients who did not develop diarrhoea. Some pro-inflammatory proteins were measured in the serum (haptoglobin, orosomucoid and a-1-antitrypsin) and increased in both groups over time, where citrulline (which may reflect a digestive tract disorder) was decreased. Comparison between the two groups showed that the measured inflammatory markers increased more in patients with diarrhoea than those without. Haptoglobin and citrulline were raised at the 5-week and 3-and 5-week time points in patients who developed diarrhoea compared to those who did not. It shows that the intestinal ecosystem is altered after radiotherapy, which seems to be even more important when patients develop symptoms such as diarrhoea [START_REF] Wang | Gut microbial dysbiosis may predict diarrhea and fatigue in patients undergoing pelvic cancer radiotherapy: a pilot study[END_REF]. The third study included 18 patients with cervical cancer. Two groups were formed among these patients:

those who developed radiation enteritis (RE) (n = 10) and those who did not (non-RE) (n = 8).

Stool samples were collected one day before radiotherapy and one day after the last radiotherapy session for each patient. a-diversity and b-diversity were measured and compared between RE and non-RE patients. RE patients showed a decrease in a-diversity (measured with the Shannon index) and an increase in b-diversity compared to non-RE patients. This result means that RE patients have either a loss of abundance of some bacteria, a loss of specific species, or both. However, the microbiota composition within this group is more heterogeneous than in non-RE patients.

The Proteobacteria phylum increased only in RE patients. Concerning the family level, three were increased in RE (Enterobacteriaceae, Phyllobacteriaceae, Beijerinckiaceae), and two were decreased (Bacteroidaceae and Ruminococcaceae) compared to non-RE samples.

Finally, within the genus level, Bacteroides, Bacteroidaceae and Plebeius taxa were increased in non-RE, whereas in RE, Megamonas, Novosphingobium and Prevotella were increased.

Unfortunately, no details were provided regarding the comparison of the abundance of all these taxa before and after irradiation in the two groups, so it is not possible to conclude that the changes in the gut microbial composition described above only occurred after irradiation (and therefore a consequence of irradiation) or that they already existed before (and thus were not caused by irradiation). A comparison with the same index was made between the composition of the microbiota of RE patients before and after radiation. It showed no difference except for the abundance of Coprococcus, which increased before radiation for RE patients. The comparison before and after radiotherapy highlights that RE patients can be detected before radiotherapy. But there is no indication of the difference before and after radiation for non-RE patients if the microbiota changes or the species diversity is different between the two groups before radiation. There is also no control in this study, so it is impossible to know whether the cervical cancer patients included in this study have a difference in specific diversity (given by the Shannon index) compared to healthy patients, as they showed in their previous study (described just before). Then the patients with RE were classified according to the grade of RE, so three grades were identified among these patients, grade 1 (n=3), grade 2 (n=3) and grade 3 (n=4), and they measured a and b-diversity. The more severe the grade, the more a-diversity was decreased, but we do not have a comparison before radiotherapy, so we cannot know if the severity grade can be predicted, as it seems that RE can. Next, the cell lines were exposed to suspensions from patients with grade 3 RE (RE3) and patients without RE. Cell cultures were exposed to suspensions from the different patient groups. On these cell lines, proteins involved in the epithelial barrier were measured. According to these measurements, patients without an RE appear to have a higher number of these proteins, indicating a less damaged epithelial barrier than RE patients. Measurements of epithelial integrity by transepithelial electrical resistance (TEER) and FITC dextran concentration were performed and showed to be decreased in cell lines exposed to RE3. Thus, these different measurements show that there would be a decrease in barrier functions and epithelial integrity in patients who have developed an RE compared to patients who have not developed one.

Concerning inflammatory markers, TNF-α and IL1b concentration (measured by PCR) were assessed in the cell lines exposed to the suspension of patients in the RE3 group. No comparison was made with cell lines exposed to suspensions from healthy patients. In addition, measurements of these cytokines were also made directly in the serum of the patients, and an increase was found for all RE patients compared to non-RE patients.

The authors conclude that the decrease in epithelial integrity and the increase in inflammation seen only in RE patients could be related to microbiota alteration (137).

Another clinical study examined the effect on the microbiota of pelvic irradiation, carried out as part of treating cervical, endometrial and rectal cancers. The researchers took faecal samples from 10 patients undergoing pelvic radiotherapy and healthy patients before, during and after radiotherapy. They examined the similarity of the gut microbiota within each group between the first sample and the other three. They found that the similarity was the same between healthy patients and patients who received radiotherapy and did not develop diarrhoea.

However, it was lower in the gut microbiota of patients who developed diarrhoea after radiotherapy. They explain that the gut microbiota may have a role to play in developing symptoms during or after radiotherapy. This study is difficult to compare with others as it did not use the same tools to analyse the microbiota, so it is not possible to compare changes in the microbiota with this study, but it is further evidence of altered gut microbiota in patients who have developed symptoms that may be indicative of the development of PRD (138).

These clinical studies have confirmed that it appears that radiotherapy leads to a change in the composition of the microbiota. It is unclear whether diversity can be increased or decreased For this section, only experimental studies involving localised pelvic irradiation were selected.

Three experimental studies tested the effect of local pelvic irradiation of the gut on host parameters such as lesions, inflammation and gut microbiota (1,[START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF]143). Each study used a different type of irradiation; the gut microbiota analysis was not performed simultaneously after irradiation, and the animals were of different sexes. The first study used 6-8 weeks old female mice and irradiated them using distal rectal brachytherapy with three to five fractions from 5.5 to 8 Gy, they also used germ-free for some experimentation, the sex of these animals was not specified (143). Gerassy-Weinberg et al., analysed the gut microbiota from faecal b. Experimental studies samples before, two weeks and six weeks after irradiation. They showed a difference in the faecal microbiota composition (analysed with 16S) between the control and irradiated groups at 2 and 6 weeks. No information about comparing control and irradiation groups before irradiation was given. The authors then showed a change in the gut microbiota composition with the b-diversity calculation between weeks 2 and 6. They hypothesised that irradiation impacted the gut microbiota between these two periods. No information about the evolution of the control group microbiota was given between these two periods. Analysis of taxa abundance change between irradiated and control mice for the times' analysis 2 and 6 weeks was made.

An increase in the phyla Proteobacteria and Verrucomicrobia but also of the genus Akkermansia, Bacteroides, Parabacteroides, Sutterella and Turicibacter and a decrease in the phylum of Firmicutes in irradiated mice compared to control were observed. To determine whether intestinal microorganisms can impact inflammation and damage to host epithelial cells after irradiation, the authors exposed cultures of colonic cells (HT-29) to a faecal suspension from mice irradiated six weeks ago and control mice. The authors then measured inflammatory markers and showed that the faecal suspension from irradiated mice increased the proportion of inflammatory cytokines produced by HT-29 cells, such as TNF-a, IL-1b and IL-4, compared to control animals' suspensions. To better understand the role of the gut microbiota in radiotoxicity in vivo, the authors inoculated GF mice with faeces from 6-week irradiated mice or from control mice. Both groups of mice were then irradiated. The authors found that mice given faeces from previously irradiated animals showed higher radiotoxicity than those from the control group. They, therefore, concluded that the faecal microbiota might increase radiosusceptibility via a process involving IL1b, which was found to be higher in GF animals that received faeces from irradiated animals. To verify that radiotoxicity in their model could involve IL1b, the authors inoculated the mice with an IL1 antagonist before irradiation.

The authors observed that the group of mice given the IL1 antagonist developed fewer lesions and showed less inflammation (measured by TNF-a) than those irradiated without the antagonist. The experiments allowed the authors to hypothesise that the microbiota could drive radiotoxicity in the rectum in a process involving IL1b (143).

The second study, performed in our laboratory, looked in part at the effect that 29Gy colorectal irradiation might have on lesions, inflammation and the faecal microbiota of rats (1). Lapière et al., used 9-week-old male rats and examined all the above parameters three days after the day of irradiation. The authors first examined the lesions and showed that three days after irradiation, epithelial damage and inflammatory infiltration were greater in the irradiated rats than in the control rats. Then the intestinal epithelial barrier was examined by measuring the concentration of HRP and FD4 passing through colon samples in Ussing chambers and showed that paracellular permeability (measured with FD4) was higher in irradiated rats than in control rats. Thus, in agreement with the results concerning epithelial damage, irradiation would appear to decrease barrier function and increase permeability. Inflammatory cells such as neutrophils and macrophages were counted in the colonic epithelium. Neutrophils were more numerous in irradiated rats than in control rats, but macrophages were not. IL1b was measured from colonic mucosal samples, but there was no difference between the group. Concerning faecal microbiota, it was measured using 16SrRNA, and results showed that neither a nor b-diversity was altered compared to control mice after irradiation. Thus, in a 29Gy colorectal irradiation model, despite the induced epithelial damage and inflammation, the microbiota is not disturbed either three days or four weeks after irradiation (data not shown) (1). While Gerassy-Vainberg et al., found a change in microbiota composition between 2 and 6 weeks after irradiation, this study did not find the same result. Although the conditions are not comparable, these two studies do not clarify the impact of irradiation on the faecal microbiota.

The third study used mice receiving 12Gy pelvic irradiation and examined the epithelial and inflammatory status of the ileum and colon and the microbiota composition in control and irradiated mice [START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF]. For the experiment, Segers et al. used individually housed 5-week-old male mice. The study was conducted from before irradiation until seven days afterwards.

Firstly, body weight was measured, and the irradiated mice lost weight from day 3 to day 5 compared to the control mice. Secondly, the state of the epithelium was measured using TUNEL assay and Ki67 staining and the measurement of crypt length. Results showed more apoptotic cells in the irradiated cells than in the control animals and as many proliferating cells.

However, the size of the colon crypts increased in the irradiated mice, which could be a marker of a repair process. However, this was not confirmed by the results concerning proliferation.

To study inflammation, the authors examined the size of the colon under the microscope, which was shorter in the irradiated mice on day three, indicating the presence of inflammation. The authors also looked at the concentration of myeloperoxidase, which only increased on day one in the irradiated mice compared to the others. The authors then examined the epithelial barrier and measured claudin-5, as well as the number of bacterial cells in the lymph nodes of the mesentery. These two measurements made it possible to identify the same time point, day 7, when claudin expression decreased and the number of bacterial cells increased in irradiated mice compared with control mice, leading to a sharp deterioration in barrier function and increased bacterial translocation. Finally, they studied the gut microbiota (with 16S rRNA sequencing). They calculated the a and b-diversity for each time point. They showed no difference in a-diversity between irradiated and control animals for each analysis time, even if, at day 7, there is a tendency to increase the Shannon index for irradiated mice (p-value =0.0524) compared to control. Concerning b-diversity, they showed that there is no difference for control animals when comparing it on days 3, 5 and 7 against the baseline. Still, for irradiated animals, there is a change in b-diversity between day 0 and day 5 and day 0 and day 7, implying that the gut microbiota has a change in this composition from day 5 onwards after irradiation and that this change continues until day 7. This study, therefore, showed that epithelial damage and inflammation appeared first after irradiation and that the gut microbiota then changed, implying that the gut microbiota is indirectly affected by exposure to irradiation [START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF].

All these studies evaluate the microbiota after exposure to irradiation at different times. It is, therefore, difficult to precisely conclude the dynamics of the microbiota alteration after irradiation. A longitudinal study would be ideal to better understand the evolutionary dynamics of the faecal microbiota after irradiation.

Only one experimental study shows results consistent with those described in the clinical studies regarding the time at which the microbiota appears to be affected by the effect of irradiation. Indeed, in clinical studies, the microbiota seems to be rapidly altered after the patients have been irradiated, which is not the case in most experimental studies. The context is different, and certain determining elements in the microbiota composition are not transposable between these clinical and experimental studies. The patients are in a tumour context in the clinical studies, which is not the case for the animals in the experimental studies.

Although it is stated that the patient's diet is more or less controlled, it is probably more diversified than that of the animals. Also, the patients treated for cancer in these studies are adults. For mice and/or rats, adulthood is considered to be reached at 12 weeks and 8 weeks, respectively, and mice and rats in these studies were aged between 5 and 8wk. The age range between patients and animals in these different studies is different, and physiological elements such as the immune system and microbiota may differ. All these parameters could influence the composition of the faecal microbiota. However, some elements are consistent, especially regarding host response, with inflammation appearing quickly after irradiation. It could lead to the hypothesis that it could be a driving force for modifying the microbiota. Still, it is impossible to confirm this with these studies alone.

PRD is a disease that can be transient or chronic; all the studies described here are in the context of transient PRD, so although the microbiota seems to be impacted in the short term, it would be interesting to know if it is also affected in the long term and the same goes for host parameters. A longitudinal study could be one more element in understanding the development of this disease and the link between the host and the microbiota that could explain it.

The common message from almost all these studies is that irradiation seems to alter and modify the composition of the faecal microbiota, either directly or indirectly, through host modification. In a context where there is no curative treatment for patients who develop PRD, the microbiota could be a good element to act on in curative treatment.

III. The microbiota as a treatment

As mentioned earlier, the clinician's primary goal when treating a cancer patient is to improve the balance between the benefits and risks of treatment.

Some aspects of radiation therapy protocols have been proposed to reduce toxicity while improving efficacy or at least maintaining it. A Cochrane review looked at some things to consider in reducing the risk of toxicity to healthy organs. It reviewed 92 randomised controlled trials and classified them according to the type and degree of radiation-induced symptoms.

They reviewed studies regarding the technique of radiotherapy delivery. They found that 3D-CRT and IMRT resulted in less toxicity than conventional RT, such as brachytherapy, in the context of endometrial cancer treatment. They also looked at other elements of the delivery technique, such as hydrogel injections or spacers. They found only low-certainty evidence of less toxicity (acute and late) in men who received this technique for prostate cancer treatment.

Some studies have suggested that the timing of radiation therapy is crucial to improving treatment efficacy and reducing radiotoxicity because of the circadian clock argument.

Theoretically, toxicity would be lower if radiation therapy was given in the evening. In this review, evening radiation therapy showed only a low certainty of reducing acute gastrointestinal toxicity (144).

Conventional treatment A. Prophylactic treatment

There seems to be a need for more data on these topics to make better conclusions regarding acute and chronic radiotoxicity. At the same time, it is also essential to continue the search for treatments for patients already suffering from PRD.

Today the treatments available to the patient do not allow them to be cured but only to improve their symptoms. Identifying the cause of their symptoms is necessary to provide the patient with the most lasting relief. For example, among the symptoms often found in patients with PRD are diarrhoea, constipation, bleeding, faecal incontinence and obstruction. Different causes in the context of PRD have been identified. Thus, Andreyev et al. have written a guideline allowing clinicians exposed to patients with PRD to orient their understanding of the origin of the symptoms with the help of diagnostic tools and the appropriate treatment for each case described. Exemples from this guideline for specific symptoms are presented below (Table 4).

B. Curative treatment

Some treatments, such as oral sucralfate, were the subject of a Cochrane review after the publication of this guideline, which concluded that there was "moderate certainty" regarding its effect on acute gastrointestinal toxicity. For sucralfate enema, the lack of data did not allow the authors to determine whether or not it could be effectively administered to the patient.

(144).

The treatments provided to patients suffering from PRD do not seem ideal because they are not sustainable and lead them to new periods of suffering. Research on improving or discovering preventive or curative treatments is still active with different strategies. The rise of studies on the intestinal microbiota in human health has led in the context of PRD to make modulation of the microbiota one of these strategies. Today, studies on the use of elements that modulate the microbiota as a preventive or curative treatment are multiplying and giving us a better idea of their potential as a treatment in the context of PRD.

Different elements could be used to modulate the composition of the gut microbiome during radiotherapy to avoid or reduce radiation-induced damage to healthy organs. Factors that may help to drive the microbiota in a certain way include prebiotics and diet. A Prebiotic is defined as "a substrate that is selectively used by host microorganisms and confers a health benefit" (146) which implies the administration of a specific substrate that would be metabolised by the microbiota and have a beneficial effect on it and, secondly, on the host. At present, there appears to be only one study concerning the use of prebiotics in the context of pelvic radiotherapy. It is an ongoing clinical trial involving the administration of a cocktail of products with an impact on the microbiota, including prebiotics, during chemotherapy and radiotherapy to patients with squamous cell carcinoma of the anal canal (147). Regarding diet, two clinical trials involving two diets for two different types of cancer are ongoing. The first is a methionine-restricted diet because it has been established that cancer cells, if not exposed to a certain amount of methionine from the diet, undergo cell cycle arrest and cell death, which is not the case for normal cells. This study aims to increase the effectiveness of radiotherapy in

Focus on gut microbiota

A. Modulation of microbiota in the prevention of PRD the context of lung, breast and prostate cancer but also to reduce its toxicity (148). The second study investigates the impact of an anti-inflammatory diet on radiotoxicity in patients with cervical cancer. It compares the low-residue diet usually recommended for patients undergoing pelvic radiotherapy for cervical cancer with an anti-inflammatory diet containing omega-3 fatty acids, antioxidants, soluble fibre and probiotics. In this clinical trial, weight, inflammatory markers (IFN-g in the serum and faecal calprotectin) will be measured, and patients will be followed up on the severity of gastrointestinal symptoms using the CTCAE classification before radiotherapy at 3 weeks, 12 weeks and 6 months (149). A recently published study on patients' quality of life during cancer treatment (including those with pelvic cancers) found improved quality of life when patients followed a ketogenic (low carbohydrate) diet. This study is based on the questionnaire responses of patients who have decided to follow this diet on their own. No information is provided about the treatments that these patients underwent. The conditions are not ideal, but the results encourage further study in the context of the ketogenic diet during pelvic radiotherapy (150).

The World Health Organisation (WHO) defines probiotics as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (151). Recently, many probiotics (containing one or more strains) have been marketed with promises of beneficial effects for various diseases. In France, probiotics are considered food supplements and, therefore, cannot be claimed for social security coverage; marketed probiotics do not need to justify their efficacy to be sold. At the European level, if a brand wishes to be considered a health product, it must be validated by the European Food Safety Authority (Efsa). Still, no brand of marketed probiotic has been validated as a health product by this authority. It does not prevent research into their effects on various diseases.

It should be noted that research on probiotics is still ongoing and much remains to be done regarding formulation, dosage and use of the strain for the drug. Each strain could have a particular effect. Today, probiotics diversity is limited because we do not know how to cultivate all the strains of microorganisms in the human gut. The strains tested are those we know, and the strains marketed are those we know how to package. The strains tested as part of the

B. Supply of beneficial microorganisms

a. Probiotics research are not exclusively the same as those sold, even if the species is the same. In addition, as with any treatment, it should be borne in mind that experimental trials are carried out in an extremely controlled environment as far as animals are concerned. Transposition to humans is, therefore, a long way off. In the context of pelvic irradiation, very few studies (n=4) have tested the effect that a specific strain of bacteria might have in reducing radiation-induced toxicity. To measure the effectiveness of treatments, all of these studies looked at one or more elements, such as tissue quality, inflammation and microbiota.

Strain

Regarding tissue quality, studies on treatment with L.delbrueickii revealed that it maintained the number of cells in crypts and goblets in the jejunum at the same level as the control.

Regarding the epithelial barrier, bacterial translocation was measured in the mesenteric lymph nodes, liver and spleen and was reduced compared to irradiated animals that did not receive the probiotic. Similarly, in terms of symptoms, L.delbrueickii reduced diarrhoea (5 out of 10 animals) compared to irradiated animals (10 out of 10 animals)(153).

L.rhamnosus treatment decreased cell height loss, luminal migration of epithelial nuclei, goblet cell loss, mitotic activity, crypt loss, atrophy or distortion compared to animals irradiated without treatment (154).

Finally, concerning tissue quality, the study on the prophylactic effect of F.prausnitzii treatment showed that this treatment reduced structural alteration at day 3 and the size of the ulceration of the colonic epithelium at day 7. Immunostaining of progenitors/stem cells with SOX9, tuft cells with DCLK1 and proliferation with PCNA in the colonic epithelium showed an increase in cell self-renewal in the colonic epithelium after treatment with F.prausnitzii (1).

Regarding inflammation, the neutrophil infiltrate was measured in three studies after irradiation and showed that neutrophil infiltrate increased. It can be thought that taking a probiotic could upset the composition of the microbiota by its mere presence but also by releasing metabolites or other elements that could modify the physiological conditions of the colonic ecosystem. It is, therefore, interesting to see if these probiotic treatments affected the microbiota. Only 1 (F.prausnitzii) of the 4 studies looked at the microbial composition between the different groups of animals. No differences were detected between the control and irradiated animals on the third day after irradiation, and no differences were reported in the group treated with F.prausnitzii compared to the other two groups (1).

Regarding the clinical studies that have looked at the effects of probiotics in patients undergoing radiotherapy, some see a beneficial effect, particularly in terms of symptoms (such as diarrhoea); others do not(155,156). It seems wise to wait for the results of other studies to confirm or not the interest in probiotics in this context. Moreover, adding a group receiving The donors were young adults aged between 18 and 24 years. As the microbiota evolves during life, the donors and recipients may be incompatible concerning the microbiota. However, it should be noted that the patient with the highest severity in the study also suffered from hepatic encephalopathy with a Child-pugh score of 9, which contraindicated any surgical intervention.

The patient had a sigmoid vesical fistula that required surgery. Although following treatment with FMT, the patient did not improve her gastrointestinal symptoms. However, the Child-b. Faecal microbiota transfer pugh score was reduced from 9 to 6, which allowed her to benefit from surgery for her sigmoid vesical fistula.

The small size of this study and the absence of a placebo group does not allow us to confirm that FMT could be an effective treatment for chronic PRD, but it does provide food for thought as to the interest in continuing studies on this subject. (157).

Of the cases reported, the first involved a woman who suffered from diarrhoea for 17 years following radiotherapy as part of a treatment strategy for her cervical carcinoma (158). The patient's quality of life was extremely impaired by her diarrhoea, which had decreased frequency from 9 to 2 times a day. Several rectoscopies and colonoscopies were performed since 2008 (4 in total) and revealed the presence of radiation-induced stenosis in the sigmoid colon. These examinations excluded the possibility of inflammatory bowel disease. The patient tried probiotics (E.coli Nissle 1917 and Bifidobacterium bifidum MIMBb75) as well as many conventional and natural treatments which did not improve her condition. In 2018, the patient was approved for FMT. She received antibiotic treatment with rifamixin for 5 days before undergoing FMT from a healthy donor. FMT was performed by colonoscopy at the terminal ileum. After the procedure, she was prescribed loperamide and discharged from hospital with no apparent symptoms. The next day she experienced severe nausea and meteorism without defecation or gas. Two days after the onset of her symptoms, she was admitted to the emergency department where she was diagnosed with intestinal loop protrusion from the walls of the distended abdomen. The patient was treated without the need for bowel resection and discharged. Her gastrointestinal symptoms improved for 3-4 weeks after FMT, but did not return to their pre-FMT level.

It is difficult to determine the impact of FMT on the patient's bowel complications, but in the long term FMT was not sufficient to improve the patient's condition. This case study raises many questions, including the use of a broad-spectrum antibiotic just prior to FMT, which has an estimated elimination half-life of 5.6 hours (i.e. it is considered to have no pharmacological effect after 28-39 hours). The antibiotic may damage the microbial composition of the patient's sample. In addition, no information is given about the donor, other than that tests were carried out to eliminate any risk of transmission of the identified pathogens. No information is given on the time elapsed between the collection from the donor and the treatment of the patient with FMT. The use of loperamide to block meteorism is also questionable when receiving a nonautologous microbiota, as blocking meteorism may increase the chances of certain beneficial or pathogenic species increasing in abundance (158).

The second case report concerns a 45 year old female patient who received pelvic radiotherapy and chemotherapy as part of her treatment for cervical cancers treatment (159). The patient has been suffering from haematochezia for 1.5 years after her radiotherapy. During her radiotherapy, the patient already suffered from diarrhoea with a frequency of 7 to 8 times a day. The diarrhoea stopped after the end of the radiotherapy but 6 months later she repeatedly detected (5 to 6 times a day 2 to 3 times a week) that stools were red with black clots and mucus. The diarrhoea was accompanied by abdominal pain and tenesmus without fever. Her stools were examined to see if she had a specific pathogen-driving disease, but nothing was found, no tumour recurrence or metastasis was found on MRI. A colonoscopy was performed and showed that there was congestion in the mucosa of the sigmoid colon and rectum with a smooth surface and bleeding easily triggered by touching the walls. An anatomopathologies analysis showed that the patient had chronic inflammation of the mucosa with hyperplasia of Although it is difficult to compare all these studies because patients who have FMT did not receive it under the same conditions, it seems that FMT could be an interesting candidate for the treatment of chronic PRD. Its preventive use has never been explored in PRD. However, its potential to modify the recipient's microbiota could make it an element to consider in this type of treatment. The microbiota seems to be impacted in the short term when healthy tissue has been exposed to ionising radiation, perhaps by avoiding this modification with FMT the patient could be saved from developing acute and perhaps chronic PRD. In a context where the patient's quality of life is already degraded due to their cancer and the treatments they are receiving, avoiding the risk of developing new symptoms would improve their quality of life and so the chances of success of their treatment.

The main objective of this PhD project was the study of the changes over time of the faecal microbiota and its metabolic environment in a relevant model of PRD in rodents.

The first axis of the thesis project was to establish a preclinical model of localised damage in rats for the investigation of PRD. We decided to use a preclinical model after fractionated irradiations in order to be closer to the clinical protocols. The characterisation of the preclinical model at the lesion and inflammatory levels was carried out for very short time points and for the chronic lesions. Finally, this model was made at two different irradiation doses to have either a moderate lesion observed in most patients or a more severe one sometimes seen in the clinic, which can alter the patient's quality of life.

Based on this preclinical model, the second axis of the study was to investigate the modifications of the faecal microbiota over time. For this purpose, we have opted for the metagenomic shotgun technique to have the most detailed picture, quantitatively and qualitatively, of the modifications of the faecal microbiota. This study was also completed by a metabolome analysis on the same animals.

These massive data were then analysed by mathematical and statistical analytical tools appropriate to the temporal data to extract the most pertinent biological information regarding microbial species and metabolites and understand the temporalities of the modifications. All this is according to the irradiation dose and thus to the lesion severity and the speed of lesion apparition and regeneration.

Lastly, the last axis of the PhD work was to explore the therapeutical benefit of the faecal microbiota transfer to have a preventive or curative action of the radiation-induced colonic damage. Cancer is the leading cause of death in the world. About 10 million people died from it in 2020.

I. Scientific paper

Submitted in Microbiome on

In the same year, 20 million new cases were diagnosed, 28% of cancers, like colorectal, urological and gynaecological cancers, with neoplasms in the pelvic area. External beam radiation therapy (ERT) is one of the foremost important treatments in managing pelvic cancer and can cure 50% of patients. Even if ERT is an effective treatment, it can be associated with several short-and long-term side effects. These effects can be due to numerous gastrointestinal toxicities due to the exposure of healthy "risk organs" around the tumour and in the radiation field. The functional loss of gut mucosae is a direct cause of the acute intestinal complications observed in most patients treated with pelvic radiotherapy (during and up to 6 months after treatment). Ten to twenty percent of patients may also develop late effects several months/years after their treatment, characterised by chronic inflammation and excessive and pathological scarring leading to fibrosis. Radiation toxicity is accompanied by co-morbidities such as dysfunctions in nutrient uptake processes, motility dysfunction, abdominal pain or tenesmus, and for patients with the most severe grades of fistulas and intestinal perforations. In 2010, side effects related to radiation-induced toxicity led to the definition of a new disease called "Pelvic Radiation Disease" (PRD) [6]. Improving cancer treatment increases the probability of developing PRD chronic side effects with a higher prevalence of inflammatory bowel diseases (IBD) [7]. PRD can significantly affect patients' quality of life and require frequent hospitalisations.

Moreover, treatments used to care for patients are only symptomatic. The temporal dynamics of PRD physiopathology is complex and still poorly described today. A better understanding of the key actors involved in initiating and developing PRD may provide an informative biological signature for personalised medicine that may be relevant to predicting RT-induced bowel toxicity or should be considered a target for curative therapies.

For several years, the microbiota has been shown to play a key role in the response, e.g., efficiency or side effects, to anti-cancer therapies such as chemo-and immuno-therapies. More recently, prospective clinical studies assessed changes in the diversity/composition of faecal bacteria before, during or after the completion of ERT. Data showed a consistent association between bacteria composition changes and gastrointestinal complications in patients benefiting from a RT [8,9]. Nevertheless, there is a discrepancy between studies concerning the variation of the faecal microbiota diversity, which increases or decreases depending on treated cancer types, ERT protocols and techniques used, individual patient response leading or not to side effects, but also index/calculation or methods used for diversity assessment [10][11][12][13]. Despite highly varied effects, these clinical data are of major interest towards advances in acute PRD physiopathology characterisation. They suggest that compositional characteristics of the cancer patient's microbiota could be relevant to predicting ERT-induced acute bowel toxicity [10][11][12][13]. Such assumptions recently opened a field of research in involving various preclinical models supporting the potential involvement of the gut microbiota in tissue in acute response to radiation [14][15][16][17][18]. Even if it was suggested that some specific gut bacteria-metabolome networks play a crucial role in substantial protection against total body irradiation-induced acute toxicity [15], there is no clear evidence of such an effect in a relevant PRD study model. This project's objective was to study the changes over time in the faecal microbiota and its metabolic environment in a relevant PRD model. We first established a preclinical rodent model with fractionated irradiations to be closer to the clinical protocols. The characterisation of this model at the level of lesions and inflammation was carried out for very short time points and for sub-chronic lesions. We next investigated the modifications of the faecal microbiota by metagenomic shotgun sequencing over time as well as the metabolome. We used mathematical and statistical analytical tools appropriate to the temporal data to extract the most pertinent biological information regarding microbial species and metabolites and explore the modifications' temporalities. Finally, using step-by-step functional analysis of microbiota or metabolome or both, as well as mathematical tools to find correlations with clinic tissue injury, we identified the functional pathway related to glutamate-ornithine-arginine-proline axis metabolism as the most relevant, that could explain the dynamics of the radiation-induced colon injury in our models.

Material and Methods

Ethics statement:

The

Animals:

The rats studied are male Outbred Sprague Dawley rats obtained from Charles Rivers Laboratories (L'Arbresle, France). Upon arrival, the rats were 13 weeks old. They were left for one month in acclimatisation in the animal house monitored environment (temperature 21°C and 50% humidity) under a reversed light-dark cycle (dark from 8 p.m to 8 a.m) before the beginning of the manipulations.

Two protocol schemes were used in this work, summarised in Supplementary Fig. S1. The first protocol aimed to characterise the histological lesions over time points, i.e., D14 and D28 postirradiation (last fraction). The 2nd protocol advised studying both the metagenome and the metabolome over time with 8-time points and the tissue lesions at D56 (the final time point and euthanasia).

Irradiation:

Rats were irradiated under anaesthesia (with a continuous flow of 1.5% isoflurane in oxygen) on a medical linear accelerator (Elekta synergy®), delivering 4MVp X-rays (mean photon energy of about 1.3 MeV). Reference dosimetry measurements were performed using a 0.125cc cylindrical ionisation chamber calibrated in dose to water in an equivalent tissue phantom placed on a plexiglass support. A dose rate of about 2.5 Gy/min in dose to water was used. The uncertainty of the dose rate measurement was about 5% at k=2. The animals were positioned to irradiate only the colorectal area (2x3cm window). The window was performed using collimation with lead bricks. To be close to the irradiation protocols found in the clinic, fraction dose irradiation was chosen. The selected doses are 30Gy and 42Gy, each delivered in three fractions of 10 and 14Gy (3x10Gy and 3x14Gy), delivered every two days. Using a fractionated irradiation protocol seems to generate mechanisms involved in the pathophysiology of the radiation-induced colon (notably the inflammatory process) close and comparable to those involved in PRD.

Samples:

The animals were euthanised at D14, D28 and D56 after the last fraction of irradiation by excessive anaesthetic inhalation. Three centimeters of distal colons were removed, longitudinally opened, fixed in 4% formaldehyde, and embedded in paraffin. Concerning the faeces samples for microbiota, the animals were collected 8 times: D0, D3, D7, D14, D21, D28, D42, and D56.

Histology:

Paraffin-embedded colons were longitudinally cut on a rotary microtome (Leica Microsystems AG, Wetzlar, Germany) into serial longitudinal sections of 5 µm, in 5 series spaced by 250 µm (all damaged zone), deparaffinised in xylene, hydrated and stained with hematoxylin-eosin saffron (HES). The severity of histological damage to the mucosa was first assessed by morphometric analysis of the crypt along the lesion with the Histolab® software (Microvision Instruments, Lisses, France). Grades of crypt damages (i) assigned were grade 0 for no damage, grade 1 for a slight dystrophic area with crypt morphological atypia characterised by disorganised and deformed epithelial crypts and/or some crypt loss, Grade 2 for high dystrophic areas from severe morphological crypt atypia/necrosis and oedema to erosion and Grade 3 for fibronecrotic areas from high necrosis/ulceration combined or not with pathological scaring (extracellular matrix deposition). The Length of the different grades (in µm) along the lesion was measured for all rats (5 series/rat) of each group. The histopathological score was performed according to Salem et al. parameters [19] with some adaptation related to specific effects induced by radiation [16][17][18][19][20].

Parameters include mucosal crypt damages and colitis, including mucosal 1) inflammatory cell infiltration, 2) submucosal inflammatory cell infiltration, submucosal oedema, colitis cystica profunda and extracellular matrix deposition. Score levels of mucosal crypt damages correspond to grade levels 0 to 3(see (i)). Colitis scores for each parameter are 0 for no effect, 1 for slight impact, 2: for moderate effect, 3 for high effect and 4 for severe effect. The mucosal crypt damage score was calculated considering each grade's proportion (length) within the lesion total length, normalised to the maximum lesion total length measured after irradiation.

The five colitis parameters measured for each rat were summed to obtain the total colitis score, which was a maximum of 20. Summing mucosal crypt damage and colitis scores calculated the total injury scores. Scores were calculated for each animal group, and data were represented using boxplots.

Staining of mast cells was realised to see the kinetics of immune cells over time after irradiation for the two irradiation doses. The slides were stained with Toluidine blue and had counterstain with Nuclear Fast Red by (Vector ref: H-3403) with an automation (PRISMA).

Immunochemistry (IHC):

IHC labelling aims to observe the appearance of neutrophils, macrophages, and the colonic epithelium over time as a function of the irradiation dose (3x10Gy or 3x14Gy). Dewaxing and treatment with 0.1% Triton and 3% H2O2 were performed. This was followed by three 5 min heat unmasking with 10mM concentrated citrate buffer at pH 6 for the neutrophil protocol. For macrophages, unmasking was done with Proteinase K (Dako S3020). The sections were circled and saturated with a blocking solution for 15 min at room temperature under agitation. The primary neutrophil antibody was a Rabbit anti-rat MPO (Abcam #ab9535).

Metagenomic

For protocol 2 (Supplementary Fig. S1), 4 faeces from all rats, at the two doses and for 8 time points, were collected and corresponded to 288 samples: 3 groups with 12 rats /group and 8 time points.

Extraction and Sequencing: Genetic material from the samples was extracted using the procedure described in previous work [21]. Total faecal DNA was shotgun sequenced using ion-proton technology (Thermo Fisher Scientific), yielding 22+/-1 million reads.

Post-processing: Reads were cleaned for low-quality reads and host information by mapping on the rat genome (mRatBN7_2) and were rarefied at 20 million reads to account for differences in sequencing depth. High-quality microbial reads were mapped on the rat gut microbiota catalogue [22] via the internal METEOR pipeline [23]. Alignments were performed using the Bowtie2 algorithm [24] with a 95% identity threshold to account for intraspecies variability [25]. Reads mapped to a unique gene in the catalogue were attributed to their corresponding genes. Then, reads mapped with the same alignment score to multiple genes in the catalogue were attributed according to the ratio of their unique mapping counts to the captured genes. The gene count table was further normalised for gene length using the R package MetaOMineR [26].

Microbial species association: Metagenomic Species (MGS), which consist of a cluster of coabundant genes, were previously built on the rat gut microbiota catalogue with MSPminer [22].

Taxonomic annotation of MGS was performed by aligning all their genes to representative GTDB r207 genomes [27] using blastn [28] version 2.10.1, task = megablast, word_size = 16.

The top 20 results for each gene were retained. A species-level assignment was given if > 50%

of the genes matched a representative GTDB genome with mean identity ≥ 95% and mean gene length coverage ≥ 90%. The remaining MGS were assigned to higher taxonomic levels (from genus to superkingdom) if more than 50% of their genes had the same annotation. The abundance of MGS was computed as the mean abundance of its 100 'marker' genes (that is, the genes that correlate the most altogether). If less than 50% of 'marker' genes were seen in a sample, the abundance of the MGS was set to 0.

Functional annotation: Genes from the rat gut microbiota catalogue were annotated on three databases of gene orthologues: EGGNOG [29], KEGG [30] and TIGRFAM [31] Modules are then reconstructed for each MGS from the genes annotations. A functional module/MGS pair is present in a sample if more than 90% of its component genes have been identified. The abundance of a functional module in a sample represents the sum of the abundances of the MGSs carrying this module.

PCoA: The Bray-Curtis and Jaccard distance matrix was calculated from the abundance table with the samples in rows and the species in columns with the "vegedist()" function of the "vegan" R package. The coordinates used for the PCoA figure were calculated with the "pco()" function of the R package ecodist. Only the first two vectors of this function were selected, and the percentages shown were calculated from the output values of the "pco()" function. Triangle points on PCoA represented the median of each group. The Bray-Curtis distance table was sorted by group and time to obtain the distance within a group for each time and represent it on the box plot with "geom_boxplot" function from the R package ggplot.

Metabolomic

For protocol 2 (Supplementary Fig. S1), faeces from all rats, at the two doses and for 8 time points were collected and correspond to 288 samples: 3 groups with 12 rats /group and 8 time points. Metabolomic was performed in the same animals as for Metagenomics.

Faeces preparation for metabolomic analysis: 25 mg of faeces were homogenised by 150 mL cooled methanol (-20 ° C) using a ball mill (MM400 Retsh® Technology, Eragny, France) for 1 minute with a frequency of 50 Hz. Samples were vortexed and incubated at -20 ° C for 30 minutes. Samples were centrifuged for 15 minutes (11000 x g, 4 ° C). The supernatant recovered from each sample was filtered through 10 KDa filter tubes (VWR, France 10K) by centrifuging for 45 minutes (11000 x g, 4 ° C). The extracts obtained were then dried using a stream of nitrogen and then frozen at -80 ° C before analysis.

LC/MS metabolomic analysis: All the dried polar extracts of faeces were first reconstituted with 125 μL acetonitrile/water (90:10; v/v). The samples were separated using an Ultimate 3000 ultra-performance liquid chromatography (UPLC) (Thermo Scientific) coupled to a Q-Exactive Plus quadrupole-orbitrap hybrid high-resolution mass spectrometer (HRMS) equipped with a heated-electrospray ionisation source (H-ESI II). Chromatographic separation was performed on a binary solvent system using a reverse-phase C18 column (Hypersil Gold, Thermo Scientific, Illkirch, France, 100mm × 2.1mm, 1.9µm) at 40°C at a flow rate of 0.4 mL min-1 and a HILIC column (Merk, France, SeQuant® ZIC®-HILIC, 150 mm × 2.1 mm, 5µm, 200 A) at 25°C at a flow rate of 0.25 mL min-1. The injection volume for both columns was 5μL. The mobile phase consisted of a combination of solvent A (0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile, v/v). The following gradient conditions were used: 0 to 1 minute, isocratic 100% A; 1 to 11 minutes, linear from 0 to 100% B; 11 minutes to 13 minutes, isocratic 100% B; 13 minutes to 14 minutes, linear from 100% to 0% B; 14 to 16 minutes, isocratic 100% A. The separated molecules were analysed in both positive and negative ionisation modes in the same run. The mass spectra were collected using 35,000

full-width at half-maximum (FWHM) resolving power for the theoretical mass-to-charge ratio (m/z) of 200. Full scan mass spectra were acquired in the 80-1000 m/z range. The ionisation source parameters for positive and negative ion mode were as follows: capillary temperature 320°C, spray voltage 3.5 kV, sheath gas 30 (arbitrary units), auxiliary gas 8 (arbitrary units), probe heater temperature 310°C, S-lens RF level set at 55 v. MS/MS experiments were performed using higher-energy collision-induced dissociation (HCD), and the normalised collision energy (NCE) applied was ramped from 10% to 40%. A quality control sample (QC) was formed to ensure good analysis repeatability by pooling a small aliquot of each biological sample. The QC sample was analysed intermittently (1 out of every 4 samples) for the duration of the analytical study to assess the variance observed in the data throughout the sample preparation, data acquisition and data pre-processing steps. Data processing and molecule identification: After LC/MS acquisition, raw data files of the entire scan -MS1 in both positive and negative modes -were converted into mzXML files.

The data was processed using the R package XCMS [32] as described previously, to generate a data matrix of deconvoluted ions with m/z, retention time and intensity. The XCMS matrix was annotated for metabolomics analysis using an internal database of more than 1300 reference compounds (based on exact mass and retention time and MS/MS spectra when detected) [33]. The annotated metabolomics datasets were further processed by probabilistic quotient normalisation to prevent sample-to-sample variation in the MS intensity response.

Enrichment analysis:

Metabolites of each multiscale analysis were put on enrichment function on MetaboAnalyst.

Each pathology selected has a p-value <0.05 corrected with False Discovery Rate (FDR).

Pathway analysis:

The involvement of metabolites in biological pathways and functions was evaluated with the specific pathway module of MetaboAnalyst using the KEGG PATHWAY [34]. Each pathway selected has a p.value < 0.05.

Statistical analysis:

Statistics and figures were made using R v 4.2.2.

Metagenomic analyses were performed using the R package momr [26]. Heat maps were visualised with the ComplexHeatmap package [35]. For beta diversity analysis, statistics were performed from the Bray-Curtis distance matrix with the "adonis()" function, which used permutational multivariate variance analysis. The number of permutations was set to 100000.

The p-values were corrected with the Benjamini and Hochberg method. Statistical significance was considered when the corrected p-value was < 0.05. The figure was created using the "geom_boxplot()" function of the R package ggplot.

The Shannon index was calculated from the species abundance table with the individual samples in rows and species in columns with "diversity()" function of R package vegan. All columns with zero or near zero sum were removed using the "nearZeroVar()" function of the R package caret before calculating the Shannon index. The figure was created using the "geom_smooth()" function of the R package ggplot. blue (e.g., CTR: blue, 3x10Gy: yellow, 3x14Gy: red).

Smoothing and clustering the multi-scale time-course profiles

The microbiota, functional modules and metabolites profiles at each time point (D0, D3, D7, D14, D21, D28, D42, and D56) can be viewed as a noisy discretisation of a continuous timedependent process:

𝑓(𝑡 ! ) = 𝛼(𝑡 ! ) + 𝑅 ! × 𝛽(𝑡 ! ) + 𝜀
Where 𝑓(𝑡 ! ) represents the opposite of the measurement of interest (for example bacterial abundance) at the 𝑖 "# time point 𝑡 ! , 𝑅 ! is a dummy variable indicating the type of dose fractionation (3x10 Gy or 3x14Gy), 𝛼 and 𝛽 a smooth functions and 𝜀 is an error sampled from zero expectation distribution. These smoothed time profiles were estimated by penalised spline functions using the R (v4.1.2) package refund. Thus, the function 𝛽(𝑡 ! ) can be viewed as a log-fold changes profile (LFC) between the irradiation and control values at the time point 𝑡 ! . These obtained LFC profiles, reduced to zero on their non-significant part, were rescaled between -1 and 1 and clustered using a hierarchical ward-linkage algorithm using the Euclidean distance. This approach (smoothing and clustering) was used as a multi-scale integration tool of the profiles of microbiota, functional module and metabolites time profiles highlighting distinct radiation-response trends.

Association between bacterial abundance/ functional module /metabolites with clinical parameters

The association between the time course of functional bacterial modules and the proportion of mucosal lesion grades or with inflammatory response enumeration at day 56, was investigated through a scalar on function regression [36,37]. More precisely, the general formulation of a scalar on function regression model is given by:

𝑦 $%& = 𝜌 ' + 8 𝜌(𝑡) × 𝑓(𝑡) 𝑑𝑡
Where 𝑦 $%& is the outcome of interest (lesion severity at day 56), 𝜌 ' is the intercept, 𝑓(𝑡) is the bacterial, functional module or metabolite time kinetic predictor and 𝜌(𝑡) is the functional parameter of the model providing an abundance weighting over the whole time frame (D0 to D56). For the interpretation, a positive (negative) value of the functional correlation parameter 𝜌(𝑡) can be interpreted as a positive (negative) time correlation between the proportion of mucosal lesion grades and the bacterial module abundance. The scalar on function regression was conducted using the the R (v4.1.2) package refund. The obtained functional parameter functions where clustered using the same approach presented in the previous section to describe the different time correlation schemes with the bacterial abundance.

Results

Development and characterisation of a relevant PRD model.

We first needed to develop and characterise a relevant model to understand the acute implication of the symbiotic relationships of the microbiota and the host's colonic ecosystem in initiating PRD. We chose to apply fractionated and localised colorectal irradiation. To validate this procedure and model relevance, the impact of a total irradiation dose of 30 or 42

Gy delivered with 3 fractions of 10 or 14 Gy was explored for the temporal dynamics (at D14, D28 and D56) of clinical symptoms and their correspondence with host biological parameters, e.g., tissue and mucosal histological damages as well as inflammatory infiltration.

Fractionated irradiation in the colorectal area induced dose-dependent host disorders and damages.

Clinical symptoms were assessed to determine the functional state of the intestine. To explore this, stool states were observed regarding colour and hydration level (symptom 1, motility status). In addition, mucus (symptom 2, epithelial barrier status/need for protection) and/or blood (symptom 3, inflammation/ulceration leading to bleeding) in faeces was noticed. The results are reported in Supplementary Fig. S2. Compared to control animals for which no clinical signs were reported over time, a few symptoms were observed after 3X10Gy irradiation and only in the long term. Indeed, 56 days after irradiation, we observed that only two animals out of 12 suffered from one of the different clinical symptoms (symptoms 1 and 2). The 3X14Gy irradiation dose induced short term clinical symptoms, mainly symptoms 1 and 3 in 7 out of 12 animals, between days 0 and 14, with a maximum peak observed on day 7. For the later time-points, all the clinical symptoms were observed in all 3X14Gy animals. We observed that 6/12 animals showed only one symptom, 4/12 two of the symptoms and 2/12 showed all three symptoms (Supplementary Fig. S2). In summary, clinical symptoms assessed after irradiation were dose-dependent in frequency, temporality, and intensity.

To take this analysis further, the state of the physical barrier integrity of the colonic epithelium at the histological level was discussed regarding morphometric analysis to quantify both the severity and the extent of structural crypt damages. Three zones were differentiated according to crypt epithelial damage states from low (grade 1, low crypt atypia) to high (grade 3 ulceration) histological alterations (Fig. 1A). The analysis of the total lesion size (sum of the size of each grade) showed a temporal dynamic represented by a so-called "bell-shaped curve" which is similar between the control and irradiated groups. Compared to the control group, after both irradiation protocols, the size of the total lesion increased significantly and similarly for the earliest times (at D14 and D28), with a maximum peak observed at day 28 (Fig. 1Ba-b-d-e and D). The maximum increase in total lesion size observed on day 28 was attributed mainly to grades 1 for the 3X10Gy group and 2 and 3 for the 3X14Gy group (Fig. 1E to G).

Compared with control values, the increase in the total lesion size persisted at day 56. However, values returned to those measured at day 14 for the 3X10Gy irradiated group and were even significantly lower for the 3X14Gy group (Fig. 1Ba-c-d-f andD). The reduction in total lesion size observed for both radiation doses at day 56 compared to day 28 appeared to be a signature of a tissue repair process that had taken place over time. The progressive reduction over time of epithelial damage in the highest grades supports this hypothesis (Fig. 1G). Indeed, even if the regenerative process seems to start after the radiation-induced acute injury phase, to become evident at the latest analysis time (D56), damages to the epithelial crypts persists, always resulting in a dose-dependent effect. We observed that 100% of the animals receiving 3X14Gy dose still having ulcerated areas vs none for those subjected to 3X10Gy dose. This finding was objectified using the lesion score, which lesion intensity normalized to the total lesion size for each animal in each group (Fig. 1H).

Fractionated irradiation of the colorectal area results in dose-dependent inflammatory responses.

To analyse radiation-induced gut mucositis, we used and performed a clinical score of the colonic inflammatory state (Fig. 2B) based on the observation of histological sections of mucosal and submucosal inflammatory cell infiltration, submucosal presence of colitis cystica profunda that is consistently described as following high severe inflammatory response, submucosal oedema and submucosa extracellular matrix deposition. For the control animals,

we observed a baseline level of the inflammatory state, mainly linked to a low and very localised increase in the parameters measured within the colon (excluding colitis cystica profunda). The inflammatory score measured was close to 0. After a 3X10Gy irradiation, we reported a sligh increase of the clinical inflammatory score. This increase progressed over time, with a maximum measured at day 56. After a 3X14Gy irradiation, the clinical inflammatory score was strongly increased compared to 3X10Gy and control groups, respectively, and maintained at this high value over time. Colitis cystica profunda was only observed after the protocol 3X14Gy at days 28 and 56, suggesting an early high inflammatory status in such irradiation conditions. We then looked at the temporal profile of the early inflammatory tissue infiltration of immune cells involved in the innate response in the colonic mucosa, i.e., neutrophils/macrophages in the lamina propria, and mast cells in the submucosa, after cell-specific immunolabeling (Fig. 2A-C-D-E). In the control group, the number of different innate immune cells quantified was very low. It remained stable for the earliest times (D14 and D28), and we observed a small decrease for the latest time-point. In response to 3X10Gy irradiation, the number of neutrophils was not modulated over time compared to the control group (Fig. 2A-C-D-E). The macrophage level increased strongly but transiently on day 14 (Fig. 2A-D), while the level of mast cells (Fig. 2A-E) was only very slightly but significantly increased from day 56. In our model, macrophages appeared to be the innate immune cells primarily present in the acute inflammatory response. It seems that after 3X10Gy irradiation, the temporal inflammatory response occurs in two phases, with a very early phase characterised by macrophage infiltration followed by a later or sub-chronic phase characterised by a strong presence of mast cells. After 3X14Gy irradiation, we showed that all types of innate immune cells significantly increased at day 14 compared to the control group.

Fig.1 Temporal effect of 3X10 Gy and 3X14 Gy fractionated colorectal irradiation on histological damage severity of the colonic mucosa (days 14, 28 and 56 post-irradiation). The study was performed 14, 28 and 56 days after irradiation. A-C Colonic sections were stained with hematoxylin, eosin and saffran (HES). A Representative pictures showing parameters considered for histological morphometric analysis, (a) grade 0 for no crypt damage, (b) grade 1 for the slight dystrophic area with small crypt morphological atypia and/or crypt loss, (c,d) grade 2 for the high dystrophic area from severe crypt morphological atypia/necrosis to erosion and (e,f) grade 3 for fibronecrotic areas with high necrosis/ulceration. B Sections show histological structure change of the colonic mucosa in one representative rat of each group and study time (scale bars 5000 µm, the total length of the lesion indicated by the black line) and C x2,5 magnification of an area of each section of

F E G H
This increase reduces over time for neutrophils, although it remains significant compared to the control group at day 56 (Fig. 2A-C-D-E).

Regarding macrophages, the temporal profile was organised into two phases. The increased level on day 14 was no longer observed on day 28 and reappeared on day 56 (Fig. 2A-D).

Finally, for mast cells, we observed a progressive increase in their number over time, with stabilised levels compared to that measured in the control group at day 56 (Fig. 2A-E). Thus, as for 3X10Gy, at the 3X14Gy dose, the temporal inflammatory response was also organised in two phases, each involving all the different innate immune cells, but with varying intensity levels of the inflammatory infiltrate. The earliest phase (D14) corresponds to a severe inflammatory response which, over time, becomes more moderate, probably corresponding to the initiation of a second sub-chronic phase.

To conclude, in our model, irradiation induced, in a dose-dependent manner, two phases of a colonic innate inflammatory response, the early phase (up to D14) and the sub-chronic phase (from D28 to D56). Thus, our model's temporal analysis window seems to represent the acute phase of PRD. We showed that fractionated colorectal irradiation leads to temporal host response with an early high-grade lesion and inflammatory phase followed by a second phase in which lesion and inflammation are persistent but of lower grade. We reported radiationinduced dose-dependent differential degrees of host digestive toxicity. Faecal microbiota evolution after a fractionated colorectal irradiation.

Fig.2 Temporal effect of 3X10 Gy and 3X14 Gy fractionated colorectal irradiations on innate inflammatory response in the colonic mucosa (days 14, 28 and 56 post-irradiation). A Representative pictures of colonic sections immunostaining by MPO (for neutrophils), CD68 (for macrophages) or of staining by toluidine blue (for mast cells) in control (CTR) animals, 3X10 Gy or 3X14Gy irradiated animals at days 14 and 56. B-E Boxplots show B colitis score including 1) mucosa inflammatory cell infiltration 2) submucosal inflammatory cell infiltration, submucosa oedema, colitis cystica profunda, and extracellular matrix deposition

Shotgun sequencing and metagenomic analysis were performed to assess potential modifications after irradiation of the faecal microbiota.

Interestingly, the mapping percentage of newly generated DNA sequences onto the reference gene catalog was about 60% (Supplementary Fig. S3A), and we observed a proportion of host DNA consistent with ulceration and severe tissue damage over time and dose. (Supplementary Fig. S3B). We first calculated microbial alpha diversity indices using the Shannon index, which corresponds to the diversity within a sample, including both abundance and richness (Fig. 3A-B). No significant differences in the alpha diversity were measured over time for either group.

On the other hand, the comparison between irradiated groups with the control group showed significantly increased alpha diversity at days 7 and 14 for the 3X14Gy dose (Fig. 3A, p≤0.05, both times). Beta diversity was then further calculated based on Bray-Curtis distance illustrates the degree of similarity between samples, taking microbial composition into consideration. The obtained distance matrix was highlighted by PCoA representation (Fig. 3C) and in boxplots (Fig. 3D-E) to study time and dose effects. The PCoA representation clearly showed temporal modification of the microbial composition (Fig. 3C) with a reduction of Bray-Curtis distance over time vs baseline microbiota in all groups but with a more rapid onset for the two irradiated groups (from D7 for 3X10Gy and 3X14GY vs D14 for control, compared to D0 Fig 3E ). A time effect, appearing more rapidly after irradiation than the control group, suggests a radiation impact on microbial composition, as shown on PCoA. After 3X10Gy irradiation, the variation in the composition of the microbiota observed seems to be very low and hardly distinguishable from variation with time itself. After 3X14Gy irradiation, our results showed a transient increase (from D3 to D28, significant p≤0.05 for all time-points, D3, D7, D14 and D28) in the Bray-Curtis distance compared to that of the control group, demonstrating in this condition a specific effect of irradiation on the change in microbial composition. In this experimental setting, even if the microbial state evolved over time in each group, we highlighted an early dose-dependent effect of irradiation on its composition.

The microbial composition was then addressed at phylum, order, family, genus and specie taxonomic level. The lists are detailed in Supplementary Table. S1 for the 3x10Gy group and Supplementary Table . S2 for the 3x14Gy group, both compared with the control group. This was investigated in temporal dynamics using a linear regression model to identify the time interval, with a significant difference in the abundance of the Taxa between the control and irradiated groups. A list of species showing significant abundance variations after irradiation was given in Supplementary Table. S3 and Supplementary Table. S4 for the 3x10Gy and 3x14Gy groups, respectively. We then focused on modifying the abundance of bacteria phylum and species clustered according to temporal dynamics. Five of the six dominant bacterial phyla and many bacterial species showed dose-dependent changes in abundance after irradiation (Supplementary Fig. S4). for 3X10Gy and Cluster 3_sp and 5_sp for 3X14Gy, respectively) to gradual and persistent increase or decrease (cluster 5_sp and 6_sp for 3X10Gy and Cluster 1_sp, 2_sp and 4_sp for 3X14Gy). To conclude this part, irradiation of the colorectal area led to a significant early change in the faecal microbiota alpha and beta diversity only for the highest irradiation dose.

Focusing on bacterial species, we observed that while the early effect seemed more pronounced, variations in abundances persisted over time after 3X10Gy and 3X14Gy irradiation in a dose-dependent manner.

Faecal metabolome evolution after a fractionated colorectal irradiation

The potential variation of the metabolome was investigated using non-targeted LC-MS/MS metabolomic analysis. The comparison of either temporal impact or irradiation dose effects of the faecal metabolite composition was first evaluated based on the Bray-Curtis distance. The PCoA representation, which considers data from all groups, did not show a marked timedependent difference in the metabolite composition (Fig. 5A). Nevertheless, when measuring the significant differences in Bray-Curtis distance within each group, differential dose and time-related variations were revealed (Fig. 5B-C). In the control group, we reported a progressive reduction of the Bray-Curtis distance over time with a significant difference at the later time-point (D56 vs D0, p≤0.05; (Fig. 5C)). In 3X10Gy and 3X14Gy groups, we showed fluctuation over time with decreases or increases depending on the time point. As reported in PCoA, this result suggests an impact of irradiation on the metabolite composition. Figure 5B compares the metabolite composition between the groups over time. No differences were observed in the 3X10Gy group compared to the control group. By contrast, in comparison with the control group, the Bray-Curtis distance increased in the 3X14Gy from day 7 to day 42 (3X14Gy group vs control group were significantly different p≤0.05 for all time-points of D3, D7, D14 and D28) supporting an impact of irradiation on the metabolite composition for the higher exposure condition. The Bray-Curtis distance approach does not allow us to completely discriminate the impact of time and irradiation on metabolite composition. We then analysed each metabolite's relative quantity variation in the temporal dynamics using an extension of the linear regression model for functional data to find time intervals that show a significant difference between the control and irradiated groups. Examples are shown in Figure 5D for dopamine, sucrose, D-ornithine and amino-isobutyrate with a green line representing the significant time interval. Out of a list of 208 metabolites identified, we found 12 and 89 metabolites showing a significant difference after 3X10Gy and 3X14Gy, respectively (listed in detail in Table . S5 for the 3X10 Gy group and Table . S6 for the 3X14Gy group). For the 3X14Gy group in which the number of metabolites generated is high, modes of variation were clustered according to their temporal dynamics (Fig. 5E). We identified 4 main temporal dynamic profiles of metabolite modifications after 3X14Gy irradiation, including clusters that either showed a transient early decrease (cluster 2_met) or increase (cluster 4_met) or a gradual and persistent decrease (cluster 1_met) or increase (cluster 3_met). The changes in the relative amount of each metabolite per group were plotted as a heat map (Fig 6A). To take the analysis further, we tried to identify both treatment-related terms and pathways using the MetaboAnalyst software. Interestingly, we reported that metabolites from clusters 3_met and 4_met have already been linked with digestive pathologies such as colorectal cancers, inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS) (Fig. 6). To summaries, fractionated irradiation-induced changes in the faecal metabolites over time.

For the highest dose, enrichment analysis highlighted that 3x14Gy irradiation could lead to

metabolites clusters (89 metabolites) identified from the temporal dynamic modulation of identified from the comparison between control (CTR) and 3X14 Gy groups. Heatmaps were produced from table of relative quantities of metabolites normalised to day 0 (D0) according to this formula : DX =(DX-D0)/D0. Yellow colours indicate a decrease compared with D0 and the dark purple colours an increase. B Enrichment analysis using

MetaboAnalyst software to identify disease related terms.

Metabolites from cluster 3 and 4 are enriched with terms related to digestive pathologies like colorectal cancers, inflammatory bowel disease (IBD), unclassified IBD or irritable bowel syndrome (IBS). Pathologies were selected when False Discovery

Rate (FDR) was <0.05.

Modulation of metabolic pathways after 3X14Gy irradiation is linked to the severity of the colonic epithelial lesion

Next, the objective was to identify in silico metabolic pathways that are potentially key to the temporal evolution of radiation-induced colonic epithelial damages. The method used was first to confront hypotheses concerning involved metabolic pathways generated in silico, either from metagenomic or metabolomic data, to identify and select those which are in common.

The second part of the methodology was to link selected metabolic pathways to the severity of colonic epithelial damage using correlation analysis. The global method to in fine propose a mechanistic hypothesis is detailed in Supplementary Fig S10.

Bacterial communities showed similar temporal dynamic modifications with those of metabolites after 3X14Gy irradiation

We used a mathematical multi-scale temporal dynamic clustering model of data (irradiationinduced variations in relation to the control group) of the bacterial species abundances, in silico generated metabolomic functional modules from bacterial genome abundance data (see functional annotation or functional bacterial modules in material and method section) and measured metabolites. This approach allowed us to select, after 3X14Gy irradiation, a community of bacterial species based on their potential capacity to exert together regulations of one or more metabolic pathways and associate them with the modulation of measured metabolites according to common temporal dynamics. Eight different temporal dynamic profiles were identified by this approach (Fig. 7A). Clusters 2-3_x_scale and 5_x_scale showed transient increase or decrease in the early period, respectively, cluster 8_x_scale a transient increase in the intermediate period, and clusters 4_x_scale and 6_x_scale showed a progressive increase or decrease over time, respectively, with a peak between days 30 and 40 before starting to decline, while clusters 1_x_scale and 7_x_scale showed progressively increase or decrease starting around day 20, respectively. The list of metabolites of each cluster is shown in Figure S11. Interestingly, when we identified both disease-related terms and functions using the MetaboAnalyst software, this time using metabolites stemming from the multiscale clustering approach, we again identify several metabolites known as associated with inflammatory bowel diseases, mainly linked to cluster 2_x_scale, 3_x_scale and 4_x_scale, all upregulated in the time course (Fig. 7B).

Fig 7 Mathematic multi-scale analysis A Multi-scale temporal dynamic clustering of bacterial species abundances, functional modules from bacteria abundance and metabolites previously identified as differentially modulated for a time period in the 3X14 Gy compared to control (CTR) animals (n=12 per group at each time point). This approach identified eight different temporal dynamic profiles. B

Metabolites extracted from the multiscale clustering approach were used for an enrichment analysis using MetaboAnalyst software to identify disease-related terms. Metabolites from clusters 2, 3 and 4 are enriched with terms related to digestive pathologies like Crohn's disease (CD), Irritable Bowel Syndrome (IBS), Ulcerative colitis (UC) and unclassified IBD. C The same list of metabolites was used for an enrichment analysis to identify functional pathways putatively linked to modifying the metabolome over time. Pathology was selected when False Discovery Rate (FDR) was <0.05. Pathways were selected when the p-value was < 0.05 and are indicated with a logarithmic scale on the y-axis.

A C B

In silico modulation of glutamate-arginine-ornithine-proline metabolic pathways after 3X14Gy irradiation

Taking this analysis further, we used the pathway function of MetaboAnalytst software to identify modulation after 3X14Gy of in silico metabolic pathways generated from the list of metabolites (Supplementary Table. S7) obtained by multi-scale clustering. This approach made it possible to identify 17 metabolic pathways including, among all, arginine-proline metabolism for clusters 2-3-4_x_scale, D-arginine_D-ornithine, glutathione, and histidine metabolism for cluster 4_x_scale, alanine-aspartate-glutamate, D-glutamine_D-glutamate, metabolism for cluster 7_x_scale (Fig 7C ). A completeness analysis in silico within each of the different clusters generated previously (1_x_scale to 8_x_scale), allowed us to select using a parallel approach, bacterial metabolic functional modules, i.e., matching with the presence of appropriate communities of bacteria in the same cluster (Fig. 8). Interestingly, this method identified both new putative pathways and previously identified ones. By cross-integrating the data from the two complementary approaches, metabolic pathways of the glutamate, arginine, ornithine and proline had been selected as interesting and, going further, suggest that the bacterial glutamate-arginine-ornithine-proline metabolism pathways was modified after irradiation. Data from functional linear regression analysis showed that the level of the 4 amino acids increases in faeces over time after 3X14Gy irradiation, with different temporal dynamics for each of them (Supplementary Fig. S6). Compared to the control group, a significant increase of glutamate was reported from day 20 to 50, for arginine from day 10 to 30 and ornithine from The temporal evolution of faecal arginine and ornithine matches with in silico increase of biosynthesis functional module (through ornithine or glutamate pathways respectively) (Cluster 4_x_scale and 8_x_scale, Fig 7A and8). In silico proline biosynthesis and degradation (glutamate pathway) mirrors temporal dynamics (clusters 4_x_scale and 6_x_scale, respectively, Fig 7A and8). Interestingly, the number of species ( 11) associated with the functional modules of proline biosynthesis (cluster 4_x_scale) is higher than the number of species (2) associated with proline degradation (clusters 6_x_scale, Fig 7A and8). Moreover, among species associated with functional module proline biosynthesis, two of them showed gradually increased abundance over time from day 14 (Supplementary Fig. S7). Although, in silico decrease of proline degradation (glutamate pathway) is predicted from days 0 to 30 and could explain the increase in proline levels measured in faeces, compared to controls at this time, its biosynthesis (through glutamate) could be the principal cause for latest times.

High-severity tissue lesion at day 56 is linked to temporal dynamic changes of the glutamate-arginine-ornithine-proline metabolic pathways.

After 3X14Gy irradiation, a scalar-on-function correlation analysis was performed between the proportion of mucosal lesion grades within the total lesion at day 56 and the temporal dynamics of functional bacterial modules or metabolites (all of them i.e., independently of radiationinduced modulation). Severe, moderate and slight lesions were defined as those with a high proportion of grade 3 (ulceration), 2 (strong epithelial crypt atypia) and 1 (weak epithelial crypt atypia), respectively (Fig. 1).

Irradiation-induced in silico changes in bacterial functional modules are correlated with the severity of the lesion

No correlation was evidenced between in silico temporal evolutions of functional bacterial modules (bfm) and slight lesions. By contrast, Figure 10A shows significant correlations with increased damage severity according to 4 different temporal dynamics for both moderate (list in Supplementary Table.S8) and severe lesions (Supplementary Table. S9). We showed that 3

Fig 9 Correlation analysis between the proportion of mucosal lesion grades within the total lesion at 56 days. The temporal dynamics of A bacterial functional modules (bfm) B metabolites (met). Severe, moderate and slight lesions were defined as those with a high proportion of grade 3 (ulceration) and 2 (strong epithelial crypt atypia), respectively. Clustering analyses A show significant correlations with increased damage severity according to 4 different temporal dynamics for both moderate (Clusters A to D_bfm) and severe lesions (clusters E to H_bfm). B the temporal dynamics of metabolite (met).

Severe, moderate and slight lesions were defined as those with a high proportion of grade 3 (ulceration), and 2 (strong epithelial crypt atypia), respectively. Clustering analyses B show significant correlations with increased damage severity according to 6 different temporal dynamics for both moderate (Clusters A to F_met) and 5 for severe lesions (clusters G to K_met).

Figure 9 A

Correlation with moderate lesion (epithelial crypt atypia grade 2) at D56

Days

B

This correlation is negative for severe lesions in cluster G_bfm. A cross-comparison between correlation and multi-scale clustering studies allowed us to identify functional bacterial modules related to glutamate, arginine, ornithine and proline for which temporal evolution was correlated with lesion severity measured at day 56 after 3X14Gy irradiation (Fig. 7A,8, 9A

and Supplementary Table. S8, 9 in which bacterial functional modules related to glutamatearginine-ornithine-proline-metabolic pathways were highlighted in green).

Bacterial functional module variations of the Glutamate seem to be correlated with lesion grade 3. Interestingly, specific reduction of glutamate degradation (4-aminobutanoate pathway) for the early period after irradiation was correlated with severe (grade 3) but also with moderate (grade 2) lesions. The early decrease in proline degradation (glutamate pathway) and increase in ornithine (through glutamate) and arginine (through ornithine) biosynthesis seems to be directly correlated with moderate (grade 2) damages 56 days after 3X14Gy irradiation.

Concerning the latest periods from days 45 to 56 after 3X14Gy irradiation, cross-comparison of analysis indicated that the onset of a decrease in proline biosynthesis (via glutamate) did not correlate with moderate lesions but, on the contrary, negatively correlated with severe ulceration-type lesions (Cluster G_bfm, Fig 7A). The same observation was made for arginine (via the ornithine pathway). This supports the potential role of such bacterial metabolic pathways in radiation-induced digestive toxicity. To summaries, in silico analysis predicted that after 3X14Gy, the modulation of the glutamate-arginine-ornithine-proline metabolism into some community of microbiota bacteria could impact symbiotic relationships in the colonic ecosystem and, in turn, radiation-induced digestive toxicity. To highlight some metabolic pathway networks interacting with those of glutamate-arginine-ornithine-proline metabolic pathways, we used the pathway function in MetaboloAnalyst software. Interestingly we identified, creatinine, sarcosine, putrescine and spermidine as potentially interesting metabolites involved after irradiation on lesion development or rescue (Fig. 10). As for glutamate, arginine, ornithine and proline, all these metabolites increased after 3X14Gy irradiation (Supplementary Table. S5, Table . S6 and Fig. S6). Unlike glutamate, arginine, ornithine and proline, none of these metabolites are described as being enriched in inflammatory diseases. They are not components of metabolic pathways identified by bacterial functional module analysis. Nevertheless, these polyamine metabolites correlated with injury severity. This observation allows us to propose glutamate, arginine, ornithine and proline as amino acids metabolised by microbiota bacteria, primarily in modulating radiation-induced colonic toxicity.

Irradiation-induced changes in faecal glutamate, ornithine and proline are correlated with the severity of the lesion

Significant correlations were also reported between the severity of the lesion at day 56 and the temporal dynamics of metabolites. We identified and J_met, showing a positive (for H) or negative (for J) correlation for the latest times. By contrast, no correlation cluster includes arginine.

A temporal dynamic cross-analysis was performed between the correlation_met study and measured glutamate, arginine, ornithine and proline metabolites in the faeces. Glutamate variations, for which faecal level follows the temporal dynamics of the lesion, could be correlated with both moderate (grade 2) and severe (grade 3) lesions (maximum severity) at day 56 post-irradiation. Faecal proline increase, from days 3 to 56, could be gradually correlated with moderate (grade 2) lesions and, from the latest times, also negatively correlated with grade 3. Finally, faecal variations of ornithine could be correlated with moderate (grade 2) lesions. Interestingly, at day 56 post-irradiation, when ornithine levels returned to the control value, a positive correlation with lesion grade 3 was predicted. These predictions highlight the likely importance of the modulation of glutamate, arginine, ornithine and proline metabolic pathways in bacteria but also probably in the host on radiation-induced lesion development or rescue.

Discussion

The pathogenesis of numerous diseases may involve alteration in the commensal microbiota composition and function [38]. In the context of radiotherapy, clinical studies, although few, proposed interesting hypotheses on the involvement of the microbiota in radiation-induced toxicity. It has been shown that after radiotherapy, the more severe the response of the intestinal ecosystem (microbiota, inflammation, and barrier integrity), the more patients develop significant clinical symptoms [11][12][13]. Ding's clinical study reinforced the hypothesis of the potential involvement of alteration of the intestinal ecosystem in radiation-induced digestive toxicity and identified faecal microbiota transfer (FMT) as a promising treatment for radiation enteritis in improving patient prognosis [39]. Although very interesting, these studies do not provide a clear picture of time-dependent potential alterations of the relationships between the microbiota and the host within the digestive ecosystem after radiotherapy. This is an important point to study, to develop therapeutic strategies against PRD. The impact of irradiation on the microbiota/host relationships has been little studied in preclinical models, and even less over time. The results obtained are not comparable due to different irradiation procedures, with the majority using single-dose whole-body external irradiation [14,15,17,18,40].

In this study, we developed a preclinical model of PRD. We chose to use a model mimicking the exposure to irradiation of tissue in the repair phase, as performed in the clinic, and allowing tissue damage similar to that observed in patients developing gastrointestinal complications after radiotherapy. Thus, irradiation at fractionated doses of 3x10Gy and 3x14Gy was performed in the colorectal area. We showed that these irradiations induced host/microbiota responses depending on the dose per fraction used. The model was representative of the early phase of PRD and the 3X14Gy dose, which induces the greatest severity of the damage, relevant to the analysis over time of alterations in host-microbiota relationships after irradiation.

In our model, 3X14Gy irradiation leads to a progressive and persistent evolution of mucosal damages, with maximum severity observed during the early period followed by a repair phase.

This temporal dynamic is identical to that of the measured inflammatory parameters (inflammatory score), as shown by the early high inflammatory peak, which remains elevated over the entire time frame of our study. Irradiation led to severe innate inflammatory response, as shown by the increase of neutrophile, macrophage and mast cell infiltration within the colonic epithelium following a time-dependent expression level for each. Such an increase has also been reported in patients undergoing radiotherapy for prostate or rectal cancers. The increase of neutrophils and macrophages was observed from biopsies of the rectal mucosa, at 2 and 6 weeks during the ongoing radiotherapy for prostate carcinoma. For mast cells, hyperplasia was shown on resected rectum 6 weeks after the completion of preoperative radiotherapy to treat adenocarcinoma [41,42]. The precise role of each of these immune cells in the chronology of lesion appearance and/or tissue repair remains difficult to describe precisely [43]. This could explain the limited therapeutic efficacy obtained by the "classical" anti-inflammatory drugs. Considering the global variations of the intestinal ecosystem, including modification of the microbiota, it seems more relevant for developing innovative therapeutic approaches.

Segers's work [18] showed that over a period of 7 days after mice pelvic irradiation at a dose of 12Gy, change in microbiota composition occurs secondary to the inflammatory peak of neutrophils and during a phase described by the authors as reparative for the colonic epithelium.

Other clinical [13] and preclinical [14] works suggested that alteration of the microbiota at later times after irradiation (on the day after the last radiotherapy session and 6 weeks, respectively) may degrade the integrity of the digestive barrier by stimulating inflammation via IL1b.

Thus, we generated multi-omic data to perform an integrated longitudinal temporal study to better understand the functional state of the symbiotic relationships between the host and the microbiota in our model. Indeed, the evaluation of colonic epithelial lesions was associated with modulations of the microbiota measured in the faeces by shotgun metagenomic and those of the metabolome (to obtain a better picture of the phenotype of the microbiota) measured by Liquid-chromatography-Mass spectrometry.

Previous clinical and preclinical studies assessed microbial variation using a metataxonomic approach consisting of targeted sequencing of hypervariable regions of the 16S rRNA gene.

Shotgun metagenomics, based on sequencing all the DNA present in a sample, makes it possible to characterize the taxonomic composition of the microbiota and their relative abundances more accurately than metataxonomic. In this study, the major driving force in using shotgun metagenomic is that this approach is more suitable for accessing the genetic contribution member of a specific community of interest regarding functional genes.

So, using this approach, we first showed that, at the peak of the severity of the lesion and inflammation induced by irradiation (early period), the microbiota shows modulations with a transient increase in its diversity associated with a variation in its composition. Using clustering and completeness analyses, with mathematical and statistical tools adapted for temporal dynamics, we showed that microbial composition variation is maintained over time and characterised by markers including different time-dependent clusters of bacteria species. Based on an ecological approach that considers the bacterial community as a whole [44] and using dynamic multi-scale clustering and correlation analysis approaches, we identified in silico the amino-acid pathway related to glutamate, arginine, ornithine and proline, as modified and associated with the dynamics of mucosal lesions of the colon after irradiation. These 4 amino acids are implicated in a wide range of complex cellular processes, including, bacterial protection through cellular bioenergetics and growth, redox signaling, osmo-protection and stress resistance [45][46][47]. Indeed, during the early phase of irradiation, when inflammation is severe and microbiota diversity increases, directing this pathway within some bacterial communities towards optimal protection could reduce the deleterious effects of symbiotic relationship alteration within the colonic ecosystem and, thus their consequences on the tissue.

During the sub-chronic phase of irradiation, orientation of this pathway towards an increase in proline and ornithine [48] production by certain bacteria could promote tissue repair. Going further, our in silico analysis also proposed the glutamate metabolic pathway as a potential actor playing a role in radiation-induced toxicity. Indeed, we showed that glutamate variation measured in the faeces over time follows the temporal evolution of epithelial colorectal injury.

Moreover, results obtained by the cross-link of experimental measures and in silico analysis showed a positive correlation between glutamate variation over time and the severity of lesions.

Our analysis also describes glutamate as being enriched in inflammatory diseases. In line with these ideas, glutamate should be investigated as a potential biomarker and an actor of the severity of the colonic epithelial lesion.

A recent study showed that multiple metabolic pathways, including arginine-proline, were predicted to be involved in the early (day 3) impact of 5Gy total body proton irradiation on the intestine [40]. In the latter study, proton irradiation is used to understand the potential effects of human exposure to cosmic radiation in the context of space missions. To our knowledge, no former study aimed to measure the impact of irradiation on time course microbiota modification related to a context of medical application of radiotherapy. In our model, which offers a better analogy to the medical context than Li's study [40], the bacterial proline metabolic pathway was in silico associated with colonic epithelial lesions after irradiation.

Indeed, early proline degradation and its tardive biosynthesis are respectively positively and negatively correlated with severe lesions. Proline metabolism is fundamental for physiological microbiota functions and those of the host, and, independently of its origin, metabolism could participate in tissue regeneration. This amino acid is particularly characterised by its ability to promote wound healing of connective tissue, as demonstrated for skin, for instance [49]. The potential impact of proline on the gut regenerative process is poorly described. Some studies using diets to modify either proline intake (amino acid diet supplementation, including proline)

or proline metabolic pathways (high fructose diet, reducing proline level) in the DSS-induced colitis model, suggested a potential role of proline in the protection and healing of the colonic mucosa [50,51]. Authors reported that positive or negative effects of respectively increased or decreased levels of proline were systematically associated with changes in the microbiota.

Nevertheless, further investigations will be required to characterise, in our model, the precise temporal impact of glutamate ornithine and proline increase on radiation-induced digestive toxicity, i.e., the role of the glutamate, ornithine and/or proline in the lesions worsening or in the repair process.

In conclusion, we performed an original multidisciplinary work using a novel preclinical model relevant to understanding the temporal mechanisms involved in the evolution of the PRD.

Using multi-omic data, mathematical and statical tools and in silico approaches, we can predict functional modification in the colonic ecosystem over time. We showed that microbiota and its metabolic environment change over time, depending on the radiation dose and the lesional/regeneration process dynamics. This work identifies therapeutic strategies targeting glutamate-arginine-ornithine-proline metabolic pathways or glutamate/proline modulation, putatively via a microbiotherapy strategies either directly or indirectly.

II. Supplementary results

Patients with PRD have no curative treatment options. In this work (scientific paper p.87), we showed that the gut microbiota is involved in forming radiation-induced damage in the colon immediately after tissue exposure to ionising radiation and even in the repair mechanism after damage. Because patients do not develop the same toxicity when receiving the same dose delivered in the same way, the gut microbiota could be more or less protective against radiotherapy and/or allow more or less optimal repair if damage has occurred. It is now recognised that FMT is effective in modifying the gut microbiota of treated patients, and, as such, it is now indicated for treating recurrent Clostridium difficile infections. In addition, FMT has shown encouraging results in treating inflammatory bowel disease, particularly in remission induction in ulcerative colitis (203). Since PRD and IBD share some common features, such as damage to the digestive tissue, inflammation, and a different composition of the gut microbiota compared to healthy patients, FMT could be an interesting treatment for PRD. We aim to see to what extent FMT might be relevant in PRD by testing its effects in an experimental PRD model. However, there are two ways of testing the impact that FMT could have, preventively to prevent radiation-induced changes in the microbiota, to limit the development of lesions in the colonic epithelium. Or wait for the stabilisation of the gut microbiota and start treatment after the lesion has formed to reinforce the optimal microbiota.

We tried several protocols for this project, including preventive and curative ones.

`

Material and methods concerning ethic animals and irradiation protocol have been described

in the scientific paper (p.87).

1. Introduction

Material and methods

Autologous FMT: the faeces samples of each animal are collected the week before the first irradiation fraction. When collected, the faeces are placed in 5mL Eppendorph® tubes. A 5% NaCl/glycerol solution (called NaCl-in the following paragraph) or media from MaatPharma® is added. The faeces are mixed with the solution (NaCl-or the media) for liquid preparation.

The preparation is then filtered with sterile gauze.

Pooled FMT: faeces from FMT and the control group were sampled before irradiation, and all faeces sampled were reunited to and mixed with NaCl-or media from MaatPharma® to obtain liquid form in 50mL Falcon® tubes. The preparation is then filtered with sterile gauze.

Once ready, the samples are put on dry ice and stored in a refrigerator at -80°C.On the transfer day, the samples are thawed and administered intragastrically or by rectal enema using a gastric tube. Intragastric administration was performed in vigil. For the rectal enema, animals were anaesthetized for 10min and held in a position where their backs were slightly raised to avoid the transfer coming out.

Seven protocols have been realised during the course of this project. Protocols and results will be presented in the order from the first (01) realised until the last (07). Each animal was sampled a couple of days before receiving the first fraction irradiation dose for each protocol. Animals that received NaCl/glycerol were sampled to experience the same stress level as FMT groups even though their faeces were not retained. For the fourth protocol (04), autologous FMT was performed, and it was delivered intragastrically. This protocol had four groups of animals (control (CTR), control +NaCl-(CTR NaCl-), IR 3x10Gy + NaCl-(3x10Gy NaCl), IR 3x10Gy + autologous FMT (3x10Gy FMTa).

Each group treated either with NaCl-or FMT included of 8 animals per protocol, and CTR and CTR+NaCl-8 animals per group for a total of 48 animals. This protocol was divided into two In protocol 01, the animals were sacrificed at D14, and the lesions present in the animals were measured. This measurement found that FMTa decreased the size of the most severe lesions in the 3x10Gy protocol. The median size of the moderate lesions (atypia +) was 14473µm and 11146µm for the 3x10Gy NaCl-and the 3x10Gy FMTa group respectively (p-value < 0.05) and the median size of the severe lesions (atypia ++) was 887µm and 0µm for the 3x10Gy To see if FMT ameliorate other things apart from the size of the different type of lesions, we looked into the count of some immune cells (neutrophils, macrophages and mastocytes) in the colon epithelium (for neutrophils and macrophages) or submucosa (for mastocytes). We saw that neutrophils were the only immune cells for which FMT did not reduce the recruitment at D14 (Figure .32.C). However, because the MPO staining seemed more or less intense, we decided to create a score to measure the intensity of the staining from 0 (low intensity) to 2 (high intensity) for each sample. We counted the number of animals per score and saw that animals from CTR, CTR NaCl-and the FMTa group were less to present a score of intensity 2

Results

(1 animal for CTR, 2 for CTR NaCl-and 3x10Gy FMTa against 5 for 3x10Gy NaCl-group) (Figure .32.D). This measurement of the intensity of MPO staining indicates that even if the number is not reduced in the 3x10Gy FMTa group, neutrophils seem less activated than in the 3x10Gy NaCl-group. The number of macrophages per unit of the area was reduced from 0.087 to 0.025 in 3x10Gy FMTa compared to 3x10Gy FMT NaCl-(p-value < 0.05) (Figure .32.E).

For mast cells no difference were detected in any group (Figure .32.F)

Results concerning lesions and immune recruitment show that FMT could help reduce it in a preventive way for the 3x10Gy irradiation protocol.

The failure of FMT in the 3x14Gy irradiation protocol might be because we needed to transfer more FMT to change the microbiota and the ecosystem. We decided to increase the number of FMT for the following protocol to see if our hypothesis could be validated. Because we wanted to improve the chance of the FMT implants, we decided to test a protocol with administration every day instead of every three or four days, and we increased the number of FMT from 4 to 7.

For protocol 02, we only realised our experiment on the 3x14Gy irradiation protocol group and tried different administration times. The common point among all three protocols (02A, B and C) is the number of administrations of FMT in order to see if the increase allowed amelioration of radio-induced injuries (Figure .26). These protocols showed that the increase in FMT number did not allow to have an amelioration concerning the size of the lesion for the 3x14Gy FMTa With protocols 01 and 02 results, we constated this it would be challenging to work on a 3x14Gy irradiation protocol to find if FMT could be an efficient treatment in an experimental model of PRD. We decided to focus only on the 3x10Gy irradiation protocol to test protocols to maximise the optimisation of the FMT protocols.

For the following protocol (03), we decided to reproduce the administration protocol from the 02 by changing administration mode. We traded intragastrically administration for rectal enema one. Our assumption was that rectal administration could improve microorganism implantation.

This protocol confirmed the results we had for the 3x10Gy irradiation. Protocol in 02 experimentation, 3x10Gy FMTa group showed a size of total lesion more little than for the 3x10Gy NaCl-group (median size of 17811,97 µm for the 3x10Gy FMTa group and 19024,98 µm for the 3x10Gy NaCl-group)(p-value <0;05) as well for low atypia lesion (atypia) (median size of 17324,74 µm for the 3x10Gy FMTa group and 18286,6µm for the 3x10Gy NaCl-group) For the following protocol, we decided to try the daily FMT dosage and keep the same amount of FMT administration (four). To do that, we administered intragastrically FMT every day for four days. To compare the protocol that shows improvement of radiation-induced injury (protocol 01 and 02) with this new one, we also reproduce the FMT administration scheme on D0, D4, D7 and D11. Animals from the two protocols 04 were irradiated same days, lived in the same room and sacrificed on the same day; they could be comparable (Figure .28).

Protocol 04 did not allow us to confirm results from the precedent study despite using the same FMT dosage (protocols 01 and 03). It even showed contrary results, with animals from the 3x10Gy FMTa group presenting more injuries than the 3x10Gy NaCl-protocol, with the size of the lesion more prominent for the total lesion (median size of 21313,5 µm for 3x10Gy FMTa median size of "atypia" lesion was 3033,5 µm for 3x10Gy FMTa group and 4348,5 for 3x10Gy

NaCl-group (p-value<0.05), the median size of "atypia ++" lesion was 7302 µm for 3x10Gy

FMTa group and 9268µm for 3x10Gy NaCl-group (p-value <0.01) and median size of the total lesion was 19310 µm for 3x10Gy FMTa group and 23694µm for 3x10Gy NaCl-group (p-value<0.01)(Figure .36B). This experimentation allowed us to think the more tardive is the treatment after irradiation more the effect on the reduction of injury in the colon. Because we already saw FMT could be effective in a protocol and not arrive to reproduce it afterwards, it is crucial to reproduce protocol 05B before confirming FMT could be efficient in a curative way in our model of PRD.

Next, we had the occasion of changing the media in what we mixed faeces during their preparation. The new media provided by MaatPharma improve the probability of strict anaerobic bacteria surviving. Because these bacteria are the most common in the colon and are involved in many processes, including maintaining a mutualistic relationship between gut microbiota and host, it could only be better for our samples. It may increase the probability of reducing the size of the lesion. To compare our previous protocols, we decided to keep our administration protocol of four faeces with daily administration (protocol 06) (Figure. After a few tests with the improvement of the methodology of transfer preparation, we failed to reproduce the first experiments concerning protocol with FMT administration on D0, D3, D7 and D11. In this experiment, we had a 3x10Gy group to compare the effect the media could have. What this experimentation showed us is that FMT prepared with pooled faeces seemed to be the less efficient protocol with an increased size of "atypia" lesion compared to the three other groups (median size of "atypia lesion of 2970,22µm in 3x10Gy group, 3401,31 µm in 3x10Gy media group, 2898,74 µm in 3x10Gy FMTa group and 6194,59 µm in 3x10Gy FMTp group; p-value <0,0001, between 3x10Gy and 3x10Gy FMTp; p-value <0,01 between 3x10Gy media and 3x10Gy FMTp and p-value <0.001 between 3x10Gy FMTa and 3x10Gy FMTp) (Figure .38). 3x10Gy FMTp group showed increase size of "atypia +" lesion compare to 3x10Gy (median size of 6110,645 µm and of 9632,37µm in 3x10Gy FMTp group; p-value

A B

First FMT experiments were done before we had the results of our experiment where the gut microbiota was measured at 8 different times between D0 and D56. We decided to begin FMT protocols with preventive treatment. Our idea was to prevent the modification of the intestinal microbiota after irradiation and thus to prevent the formation of lesions or at least to prevent them from being severe. To achieve this, we treated the animal on the day of the last irradiation dose four times, either every day for four days or every three to four days until D14. We tested autologous (from the same animals that would receive it) and pooled FMT. The advantage of autologous FMT is that the microbiota knows the ecosystem where it will be re-implanted. We thought this might facilitate microbial species implantation into colonic ecosystem even if environment should have changed due to irradiation. In another way, we also tried pooled FMT, which consists of sampling several animals, mixing all the sampled faeces and dividing them into several transfers that the rats received after irradiation. The strategy is different with pooled FMT, as it does not reinstate a common ecosystem like the pre-irradiation one in terms of microbiota, but it can bring in many more species, in large numbers, that could compete with the gut microbiota present in the rat's ecosystem and, as the transfer should be more diverse, it could win this competition. As we could see the advantages and disadvantages in both cases, we decided to test them both to see if they could provide a therapeutic benefit. As we decided not to empty the luminal contents before treating with FMT, to avoid worsening damage to colic tissue (204), we had to repeat the operation several times to ensure implantation. We tried two different methods, either separated by a few days or every day, each type of protocol using the same amount of FMT, i.e., four. We only experimented with more FMT once for the 3x14Gy protocol, but we realised how stressed the animals seemed on the day of the last FMT administration, so we decided not to try it again. We also tried different routes of administration, oral gavage or rectal instillation. In animals, oral gavage is simpler than rectal instillation, it can be done without putting them to sleep and it takes less time. But rectal instillation seems to be the best way to avoid destroying the microbiota that we have implanted.

Oral feeding involves the microbiota going through a lot of different environments. To ensure that the microbiota is not altered, it seems more prudent to administer it directly to the site of

Discussion

A. Preventive treatment a. Preparation of protocols interest, i.e., by rectal instillation. We conducted protocols with both routes of administration to see if one had an advantage over the other in terms of effect. The last thing we changed during these FMT experiments was the media in which we prepared the faeces. Initially, we used NaCl glycerol to protect the bacteria by avoiding ice crystal formation. We then used a media from MaatPharma® which protects the bacteria, particularly the anaerobic bacteria which are in the majority in the colon.

We carried out the protocols and tried to improve them at every stage, so we can say with certainty that the FMT was ineffective because it was unstable in the context we used it. Only two protocols suggested that FMT administered every three or four days after the days of the last dose of the irradiation fraction, intragastrically or with rectal enema, could be effective for the 3x10Gy irradiation protocol as a preventive treatment. It is partly on the basis of these results, that we decided to work only on the 3x10Gy dose and to retest one of these protocols (only for oral gavage which worked better). The context in which both protocols were carried out differs from the others because we were confronted with an animal house problem during both of experiments. For the first one (intra gastric gavage), during a weekend, just after the first FMT, the ventilation system of the cages broke down. As a result, the animals got hot during the whole weekend. We decided to continue the experiment as planned, performed the three remaining FMTs and euthanised the animals at D14. Analysis of the animals' colonic tissues revealed a large difference in tissue quality. For the 3x10Gy groups, the tissue quality of the FMT-treated animals was significantly higher than that of the animals receiving NaCl/glycerol alone. When lesion size was measured, it was confirmed that FMT-treated animals had smaller and less severe lesions than NaCl/glycerol-treated animals. Furthermore, the staining of immune cells (neutrophils, macrophages and mast cells) revealed that immune system recruitment was lower in the 3x10Gy FMTa group. FMT seems to work in this context, but we only have replicated this result once whereas another problem occurred in the animal house. For the experiment with rectal enema, animals were private of day/night cycle for two weeks. The circadian effect seems to have an implication in the gut microbiota composition, but also with metabolic and immune change (205). With the privation of dark, animals could have been stressed and responded differently to irradiation and FMT as the "atypia ++" type lesion appears to be very tiny in these animals. Results from these two protocols suggest that stress could be a driver of the FMT effect in our experimental model.

b. Discussion of results

We did not have the results of the dynamics of the gut microbiota at the time, but when we obtained them and found that the microbiota of the 3x10Gy group did not appear to change after irradiation compared to the control animals, concerning the Shannon index and bdiversity (Bray-Curtis). If these results are reproducible, this means that all the animals irradiated with a fraction of 3x10 Gy did not have a change in their microbiota compared with the control due to irradiation, and therefore in particular the animals in the 3x10Gy groups in the FMT experiments. However, some animals were exposed to some stress (protocols 01 and 03). Their Exposure to stress for animals in protocols 01 and 03 did not lead to an increase in total lesion size. Still, it did decrease the size of severe lesions compared to all other protocols where sacrifices were made at D14 in NaCl-treated animals (Figure .39). It also allowed the FMT to work. FMT could have worked because the lesions were less severe in these animals.

But it is conceivable that these stresses led to changes in the colonic ecosystem. Indeed, the immune system could be altered in both situations (high heat induces stress and circadian disruption cause stress) and behave less operationally (206,207). Concerning the disruption of the circadian rhythm, the intestinal microbiota could also be impacted and participate in altering the immune system (208). In a situation where the lesions are less severe, and the immune system and microbiota may be impaired, FMT seems to be efficient. This would explain why we see a reduction in the lesion size for animals FMT-treated in protocols 01 and 03 and not in others.

This idea is reinforced by the literature concerning FMT in the context of total body irradiation.

After exposure to total body irradiation, the authors tested the effect that FMT could have on radiation-induced colonic lesions in animals. As already mentioned, total body irradiation affects the bone marrow and, thus the immune system, which is sterilised. In this context, the authors found a positive effect of FMT on animals regarding colon damage and the haematopoietic syndrome generated by total body irradiation (209).

Although heat stress also affected the 3x14Gy group in protocol 01, it did not allow FMT to be effective. It is difficult to compare lesion size as we did for the 3x10Gy FMT protocols as the number of FMTs was not the same between the two 3x14Gy FMT protocols where sacrifices were made at D14. The stress induced by seven days of intragastric gavage may impact the lesions. The increase in animal ulcers in protocol 02C compared to protocol 01 could be due to gavage stress and not heat stress, but we cannot conclude anything. By examining the results of the microbial dynamics after irradiation, it appears that the diversity of the microbiota in the 3x14Gy group increases, which may have contributed to the failure of FMT on these animals.

experiment and monitor inflammation by measuring faecal calprotectin or lipocalin in animals at different times after irradiation to see how it changes.

Ways to improve our protocol will be discussed later.

While FMT is not effective when administered as a preventive measure, it could be interesting as a curative measure as it was already implied in a clinical study mention earlier (157). Further experiments are needed to confirm these initial results.

Conclusion

I. Strengths and weakness of the preclinical model of PRD in rats to study colic ecosystem over time : what

this study bring ?

The initial objective of this PhD work was to study the impact of localised colorectal irradiation in a relevant pre-clinical model on the time course of the microbiota. The work is crucial to answering the following translational objective of whether faecal microbiota transfer could be an excellent candidate for treating radiation-induced colonic epithelial lesions in rats. To answer the clinical question, exploring what happens in the whole gut ecosystem after exposure to ionising radiation is necessary to know the most appropriate time to apply a treatment. This study is set in the context of PRD pathology, where patients who have just undergone cancer are suffering from a side effect of one of their treatments which is pelvic radiotherapy.

. PRD remains a poorly understood disease, although its incidence is estimated to be higher than that of IBD. In addition, the name PRD, while already quite common, is not yet used by all clinicians. Semantics pose a problem in the identification of patients with PRD. From a scientific point of view, it complicates the analysis of clinical studies and therefore slows the understanding of the disease. The pelvic region contains many organs that can be affected by different types of tumours. Thus, patients who have developed PRD may have been treated for different cancers and have received different types of radiotherapy and radiotherapy protocols (dose, volume and fractionation). The multitude of possibilities in the clinic is also reflected in the experimental setting. The complexity of moving from the clinic to the experimental setting to study the biological factors involved in PRD is more understandable. The review of the literature on experimental studies of PRD makes the first observation that there are only a few studies on this topic. In the context of our research on the host-microbiota relationship in PRD and the microbiota treatment of this pathology, researchers have already developed several models. Each study we have seen has its experimental protocol considering the type of radiotherapy, the dose and dose delivery protocol, and the location of the irradiation. It is important to note that all these studies are based on healthy tissue. Different results have been reported concerning modifying the intestinal microbiome after exposure to ionising radiation.

Irradiation protocols

Still, it should be noted that the analysis times and the experimental protocols differed between studies. With the benefit of hindsight from the studies examined and knowledge of the laboratory where the experimental protocols were developed, we will review the different choices made regarding them.

Our laboratory has a linear particle accelerator in the Megavoltage range, similar to those that can be used clinically for high-energy photon radiotherapy, with which experimental models are performed. EBRT is a frequent mode of irradiation in pelvic cancers; using it to reproduce PRD experimentally seemed relevant.

Historically in our laboratory but also by other teams, radiation-induced colorectal toxicity studies are mainly carried out with a single-dose irradiation protocol. The knowledge of the biological effect acquired under these conditions is not negligible. Despite the frequent use of single-dose protocols, developing fractional dose protocols to approximate the scheme used in clinical protocols would be interesting and more relevant. The fractional irradiation protocol mimics the exposure to tissue irradiation in the repair phase, as observed in the clinic. However, clinical protocols contain more fractions and lower doses per fraction than the dose used in experimental protocols partly because rats regenerate better than humans and can withstand higher doses in a smaller fraction. In addition, the protocols are spread over several weeks and are performed every day or even several times a day. Although close to the clinic, the experimental protocols are far from it. For this study, we decided to try, for the first time in the laboratory, a dose fraction protocol based on known single doses. The aim was to find a fraction protocol which could provoke the same biological effect as a single dose already studied by researchers in the lab. The Biologically Effective Dose (BED) (1) model was used. The BED was first introduced by Fowler JF in the British Journal of Radiology in 1989 to compare the radiotherapy effect on the tumour provoked by different schemes of radiotherapy fraction protocol.

( A. Type of irradiation

Single and fractioned dose

The use of the BED allows having a fraction dose irradiation protocol which should cause the same effect on the tissue concerned (here, rats colon) as a dose of 21 (which provoke moderate injuries) and 27Gy (which provokes severe injuries) to develop two models, one moderate and one severe. Thus, for single doses of 21 and 27Gy, the dose per fraction (calculated with the BED taking a/b at 10) was 10 and 14Gy, respectively, and the number of fractions was 3, so the two fractionated dose protocols were 3x10Gy (total dose 30Gy) and 3x14Gy (total dose 42Gy). An experiment was performed comparing the single dose to the BED equivalent fractional dose protocol. It showed that on the assessment of tissue lesion size (by lesion type)

and the number of immune cells per tissue area. Thus, despite choosing a protocol based on BED calculation, the biological effects are different between single-dose and fractionated BED-equivalent protocols. It is consistent with some results reported in the literature (210,211)

(Figure.40).
Regarding literature, only one study used a fraction dose protocol, but the type of irradiation done was brachytherapy (143). It would be the first study using external beam irradiation with an irradiation fraction dose protocol in the context of gut microbiome exposure to irradiation in rats.

172

A B C D E F
Radiation-induced gastrointestinal toxicity does not only occur in the context of pelvic radiotherapy. In a clinical setting, it can also be caused by whole-body clinical exposure to radiotherapy, whose main purpose is not to treat the digestive system but to deplete the bone marrow and sterilise the immune system. It is applied in the context of liquid cancer so that patients can receive bone marrow and/or stem cell transplants without facing rejection. In an accidental context, exposure to ionising radiation above 6Gy over large parts of the body causes the so-called gastrointestinal syndrome. This syndrome is an emergency: without treatment within hours of exposure to ionising radiation, the patient's death is inevitable within several days. In these cases of exposure to ionising radiation, different compartments other than the tissues of the digestive tract are severely affected (bone marrow and/or endothelial compartment (212), which is not the case in PRD. Thus, in experimental studies, it is possible to see some authors using total body and abdomen irradiation models to reflect the pathophysiology of PRD.

In contrast, in these models, the animals will have no immune cells (total body irradiation model) and/or very severe endothelial damage (total body and total abdominal irradiation model). In the case of PRD, the patient's immune system reacts strongly, which is not the case in the total body irradiation model, which is one of the limitations of these models.

Furthermore, the radiation doses used for total body and abdominal irradiation cannot be as high as for colorectal irradiation; otherwise, the animals die very quickly. The severity of the damage to the colonic tissue caused by this type of protocol only mimics, at best, the lesions found in very mildly affected PRD patients. For these reasons, we consider here that these models are more suitable for studying radiation-induced gastrointestinal syndrome than for studying PRD. Thus, to develop an experimental protocol, we relied on the laboratory's knowledge of a localised irradiation model in the colorectal region and models from the literature performing pelvic irradiations. As mentioned above, we decided to use a fractionated protocol. One of the disadvantages of fractionated protocols on a precise area on small animals such as rats with a LINAC designed for humans is that we can't use a CT to get an accurate view of the organ targeted by the irradiation. Thus, for a fraction dose treatment, when setting up rats for irradiation, no certainty is given on the positioning of the organ during irradiation concerning the irradiation session that precedes and/or follows. To ensure maximum reproducibility of the irradiation area, precautions were taken by marking the position of the rat's penis. Furthermore, to prevent the small intestine, which is highly radiosensitive, from entering the irradiation field, a belt was installed in rats to hold it as high as possible.

As mentioned earlier, all experimental studies involving gut microbiota and pelvic irradiation have targeted healthy tissue. It is a choice to be considered when the experimental design aims to understand better a pathology where patients have cancer. Furthermore, given what is known about patients with gynaecological and prostate cancers, some may develop gastrointestinal symptoms before treatment, indicating a degraded host state in the colonic ecosystem.

However, the link between pre-existing cancer-induced gastrointestinal symptoms and the risk of developing PRD has not yet been addressed in the literature. Nevertheless, IBD, a condition

B. Type of tissue targeted by irradiation

where the gut ecosystem is severely degraded, is considered a risk factor for developing PRD in patients (213).

Regarding the gut microbiota, the studies on pelvic radiotherapy and the gut microbiota mentioned earlier in chapter II show that the diversity of the gut microbiota does not seem to be altered between healthy patients and cancer patients who did not develop symptoms during or after radiotherapy (135,136). Regarding the composition, it appears that healthy persons, cancer patients who did not develop symptoms and cancer patients who developed symptoms have their own microbial composition, so each group has different profiles [START_REF] Wang | Gut microbial dysbiosis may predict diarrhea and fatigue in patients undergoing pelvic cancer radiotherapy: a pilot study[END_REF]138). Given these results, the gut microbiota seems stratified between healthy individuals and cancer patients (gynaecological or prostate). In recent years, knowledge of the gut microbiome has attracted researchers' interest in the cancer context. Several studies have compared the gut microbiota of healthy individuals and cancer patients. Some of these studies have been carried out in the context of pelvic (gynaecological or prostate) cancer and have shown differences in the composition of the gut microbiota between healthy individuals and cancer patients (214,215). These studies, therefore, seem to point in the same direction, but further studies are needed to confirm these results. However, at present, the studies do not consider the whole colonic ecosystem, which means no information is given on the type of symbiosis between the host and the microbiome during cancer before treatment. In this situation, it doesn't seem easy to reproduce something that has not yet been established. Added to this is the complexity of setting up an experimental model in which the tumour could be treated as it is in the clinic and cause PRD. In our study, we work only on healthy tissue, without any tumoral environment.

We are aware of the limitation that it is impossible in this model to know what happens when the colonic ecosystem, in the context of a pelvic tumour, is exposed to radiotherapy at the level of the host and microbiota. Nevertheless, this study provides an element of reflection on the impact of exposure to ionising radiation on the relationship between the host and the microbiota, considering that if this relationship is already degraded beforehand, it will only worsen.

The animals we decided to work on, are rats, which is not the most common rodent model in general, and this is also the case in gut microbiota research. This work aims to study what happens in an integrated system with the communication of different elements within the body, so the species used is not relevant in this respect. Rats in the context of our study have

C. Animals

advantages and disadvantages. The first advantage is that our laboratory has been working with rats for some years and therefore have a great deal of experience with the irradiation protocol and the consequences of exposure to it. The historical reason why rats were chosen rather than the mouse is that the laboratories have been working on treating mesenchymal stem cells in PRD and that mesenchymal stem cells can only be prepared from rats. Thus, the laboratory has an in-depth knowledge of the colorectal irradiation model in rats and what this can lead to.

Furthermore, to perform colorectal irradiation with a 4MV linear accelerator LINAC, and especially to realise fractionated schedules, rats are more convenient than mice because the irradiation field is extremely small and the tissue to analyse radiation-induced damages is larger. However, this model has some limitations, and the first one concerns the background analysis of the intestinal microbiota. To analyse the gut microbiota, we chose shotgun metagenomic rather than 16S rRNA sequencing, and we will discuss this choice later, but few studies doing shotgun sequencing are working on rats. The consequence is that there are few databases of microbial genes in rats, so there is a risk that more DNA sequences will not find a match in the identified microbial DNA sequence database. In our case, we expected to get 70-80% matches, and we only got 60% (Figure .41). Building a new database can take some time and, as far as our project is concerned, it is still underway at MetaGenopolis. The aim of increasing the database is to obtain the percentage of initially desired mapping (i.e. 70-80%) and, thus, a better representation of the species in the samples. Finally, although we have yet to see the benefits, this study increases the database on microbial DNA in rats. Another difficulty that we had to consider is that rats are larger than mice, which makes housing conditions more difficult. As we wanted to have many animals per group, we had to think about the distribution/number of animals per cage. We decided to house three rats together per cage, with twelve animals per group and four cages of animals per group. The risk of housing the animals together is to have a "cage effect", especially as rats are coprophagous. It increases the risk that they share a common microbiota and therefore have a common reaction to irradiation.

The other solution to avoid the bias created by the cage effect is to house the animals alone, as was done in the study by Maarten Van de Gutche et al., who sought to understand the dynamics between the host and the microbiota when rats were exposed to Dextran Sulfate Sodium (DSS) after animals were exposed to a specific regimen. For this study, two DSS doses were chosen to see if there was a step in the formation of dysbiosis and the dose effect of this. The authors performed a longitudinal study to observe the dynamics within the host and the faecal microbiota (216). In the concept, our study is very close to Van de Gutche's research, but instead of DSS, we use irradiation and do not study the impact of specific regimens. The advantage of housing the animals alone is that it avoids the "cage effect", but it could create another bias as rats are social animals, and isolation seems to create stress in them (217). One of the objectives was to reproduce the effect of a single dose (which the laboratory is familiar with) with a fraction dose protocol. Therefore, we did not want to add the risk of increasing radiation-induced lesions by our protocols because the animals were more stressed due to isolation.

Furthermore, we identified a "cage effect" at the beginning of the experiment (i.e. before irradiation). Still, this effect seemed to decrease over time in each group (Figure .42). In retrospect; we could have rearranged the groups after rats were adapted to the animal house and before irradiation, which seems to be the best option to reduce "cage effect" at the beginning of the experiment. The last thing about the animals is their sex; the study only included male animals, whereas the PRD involves both men and women, so it would have been interesting to use both sexes. It should be taken into consideration for further studies.

After discussing the irradiation protocol, we will discuss the study design for the gut microbiota analysis. longitudinal study. How much analysis time do we need, and which times would be interesting to perform this study? We answered these questions based on the support of the laboratory's knowledge of the timing of radiation-induced changes in the host and on the literature on the evolution of the microbiota after irradiation. Although there does not appear to be the same effect on the tissue between the single and fractionated dose protocols, we relied on laboratory studies showing that injury and inflammation occurred rapidly after irradiation. To see if the host and microbiota events were concordant, we decided to set the second time point of analysis (after the one before irradiation to have the baseline composition of the microbiota for each animal) close to the last fraction of irradiation, D3 after irradiation. To set the other analysis times, we also relied on studies on the evolution of the gut microbiota over time after exposure to pelvic irradiation. As the results between the studies were not the same (one study showed changes at D7 and the other at 6 weeks after irradiation [START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF]143) , we decided to set several time points between these two analysis times to make sure that we did not miss the change and to clarify when it was occurring. Thus, we decided to do 8 microbiota/metabolomic time points measurements (D0, D3, D7, D14, D28, D42, D56) with three host histological time points (D14, D28, D56). with Unifrac Unweighted and weighted distance between samples was shown to be different between the irradiated group and the control group after irradiation. However, the samples of the irradiated group at D0 were not included in this distance calculation, or at least not represented on the graph. In contrast, the samples of the control group were included. We might think the control animals at D0 included the control and irradiated groups. However, the number of animals for the 2wk and 6wk analysis points was 9, as in the 2wk and 6wk irradiated group animals. Thus, if the control and irradiated groups were pooled at D0, should be 18 animals in this group, but there were only 9 (0wk). Under these conditions, it is impossible to confirm that the difference between the groups is due to irradiation or that it was already present in the animals at D0, as we found in our samples between the 3x10Gy group and the control and 3x14Gy groups. However, they would appear to have these samples at 0wk for the irradiated group as the authors used them in a heat map that compared the abundance of some species selected by their analysis between the 0wk, 2wk and 6wk irradiated samples. This heat map showed a difference in the abundance of selected bacteria. Still, as the control heat map was not provided, it is impossible to say whether this change in particular bacteria is due to the effect of time or irradiation. The authors attribute the formation of dysbiosis between 2 and 6 weeks after irradiation, but it isn't easy to reach the same conclusion with their results (143).

For the second study, Segers et al. measured diversity a-diversity and showed no difference between the control and irradiated groups at any point in the analysis. However, calculating bdiversity with the unweighted Unifrac distance showed that the irradiated animals showed a difference in their microbial composition between day 3 and day 7 compared to day 0. These changes were not identified in the control group. These results lead to the conclusion that irradiation impacts the gut microbiota. However, it is not stated whether the irradiated animals show a difference compared to the control animals over time. The only clarification on this subject concerns the time of day 0 when the animals show differences in the microbial composition according to the group. However, the irradiated group of animals may have a different microbiota which becomes closer to that of the controls over time (as observed in our study). In this case, irradiation could accelerate the rapprochement of the microbial composition of the two groups of animals but may not necessarily have a deleterious impact on the host. Segers et al. then studied the difference in abundance between the OTUs of the irradiated group and found 25 OTUs with a difference in abundance over time. However, the authors did not investigate which species might have changed in abundance in the control group. It could have allowed us to see if some of the bacteria identified among these OTUs were strictly due to irradiation or perhaps due to a possible time effect. In addition, the authors refer to the microbial signature of irradiation in the changes in these 25 OTUs. However, we do not know if the microbiota composition of the animals in the irradiated group was similar at D0. If not, it might be interesting to consider these OTUs as a potential biomarker of microbiota damage after exposure to ionising radiation. It would mean that despite the difference at the beginning, once exposed to irradiation, the environment selects specific bacteria that would be the only ones able to grow at the expense of others that would see their abundance decrease. If the gut microbiota were the same from one animal to another at D0 between irradiated animals, OTUs could only be seen as biomarkers once other studies confirmed that each animal exposed to irradiation had the same increases and decreases in these bacteria [START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF].

This study is interesting, but it only goes beyond day 7 for analysis, too for histological or microbiota studies, which prevents us from saying whether this change in microbiota will have a subsequent impact on host parameters.

The strength of our study comes from the dynamics, as we can identify how the microbiota evolves over a long period after exposure to ionising radiation. However, in addition to the dynamics, the other strength of this study is the cross-analysis between shotgun metagenomics and metabolomics and some host parameters, which allowed us to better understand the type of symbiosis between the host and the microbiota over time after irradiation.

As the host and the gut microbiota appear to be interdependent, if a change occurs, it should be replicated in the other. Thus, studying one without considering the other could misinterpret the results regarding the attribution of responsibility in a specific disease. However, even if both compartments are subject to change, the mutualistic symbiosis may have some resilience.

Strength of studying both compartments of the colic ecosystem

When exposed to stress or environmental change, the host and the gut microbiota may show transient changes (transient alternative state) before returning to the normal state (mutualistic relationship). Pathological situations seem to occur when the changes are not transient, and the symbiosis changes from a mutualistic relationship to one that could be described as parasitism, as the host does not benefit from its relationship with the gut microbiota, whose composition and associated functions are selected by the pathological environment (stable alternative state).

However, it is still being determined how the breakdown of the mutualistic relationship between host and microbiota occurs. Understandably, when physiological conditions change, this impacts the living microorganisms that make up the microbiota. But it is essential to consider that the microorganisms in the gut are more numerous than the host cells, have a high mutation rate and could therefore cause this physiological change (218). Possibly the two events need to occur together to bring about an irreversible change in the host and the microbiota. However, it seems crucial to always consider all options when studying a specific pathology.

While it seems necessary to consider both compartments when studying the symbiotic relationship between the host and the microbiota, the question remains as to what type of analysis should be performed to be optimal. For many reasons, most research on the microbiota only considers its composition and associates it with the pathological characteristics of the host.

However, a specific species can have a different phenotype depending on its surroundings. In the case of the host, for example, macrophages can have different phenotypes depending on the environment in which they are found, either pro-inflammatory or anti-inflammatory in a simplified way. Similarly, a given species may be pro-inflammatory in one environment and anti-inflammatory in another (219). When studying the immune system in detail, the presence of certain types of cells is not the only parameter sought. The presence of the markers they express is also studied to understand their dynamics better. The principle is the same for microorganisms, so their phenotype must also be considered. One person's microbiota may differ from another in its composition but does not differ in the production of essential elements for the host, leading to a similar state of symbiosis between the two individuals. In this case, the risks are to assume a link between the microbiota and the disease that could be false.

Conversely, if the composition of the microbiota is the same between a healthy person and a patient, but the functionality of the microbiota differs, the microbiota could be considered nonharmful. In contrast, its functionality could indicate that it is (220). Since sequencing host and microbiota DNA or RNA can be extremely expensive and time-consuming, a more straightforward method would be to study the proteome or metabolome and link it to the host or microbiota. Even if this cannot be confirmed, it should give an idea of the possible dynamics.

With this in mind, we decided to carry out a faecal metabolome study on the same animals as the faecal microbiota study and carry out shotgun metagenomic sequencing instead of 16S rRNA to try to link the potential functions of the bacteria measured with the metabolites measured in each sample.

Thanks to the metabolomic and metagenomic parallel study, we observed no difference in gut microbiota composition and metabolome between 3x10Gy and control groups. However, we could identify metabolome differences in the 3x14Gy group from controls on D56. In contrast, the b-diversity of the microbiota was similar at this time, which could mean that either the host is the only one still affected by the irradiation exposure at D56 or the gut microbiota could still be affected by this exposure and participate in the metabolome modification.

As indicated in the scientific article (p.87), we examined the dynamics over time of the metabolites, species, and functional modules in the 3x14Gy group compared with the control group. We highlighted 8 different profiles, called cluster_1-8_xscale clusters, including all the metabolites and functional modules showing differences between these two groups over time.

Some of these clusters showed different dynamics at D56 between the 3x14Gy group and the control group. After investigating whether specific metabolites could be linked to the functional module of the bacteria, we found metabolites from the arginine-proline pathway in particular glutamate and proline. We established that the alteration in this metabolism could be partly linked to the microbiota, even at D56, whereas its composition did not differ from the control for the 3x14Gy group. These results contribute to the hypothesis that even if the microbiota is not different between two living organisms in terms of composition, its phenotype and, therefore, its functionality may differ. In this case, some of these metabolites (possibly derived from the microbiota) at D56 could be positively or negatively correlated with the extent of severe atypia or ulceration at that time in animals. As mentioned in the scientific paper, glutamate is linked to the severity of the lesion. In contrast, proline biosynthesis is related to a moderate lesion and appears protective in the final phase of the analysis (scientific article p.87).

In our study, we highlight one pathway related to microbiota that is correlated with the host.

We have to consider there are probably others. We only study faecal metabolites. However, all metabolites are not eliminated only within the faeces. For example, the superoxide dismutase, described as an antioxidant, is identified as a functional module of bacteria in our samples of 3x14Gy group animals. According to the completeness study, 1 functional module of superoxide dismutase is associated with the dynamic of the cluster_7_x scale, which decreases over time.

Furthemore, the decrease of functional module superoxide dismutase positively correlates with severe atypia and ulceration. However, superoxide dismutase is an enzyme whose activity is measured by western blot, activity gel or activity assay (221). As superoxide dismutase is found in the cell, tissue is the most appropriate sample for measuring activity. However, it is possible to detect oxygen-reactive metabolites such as malondialdehyde or F2-isoprostane using techniques such as LC-MS. Malondialdehyde and F2-isoprostane are products of lipid peroxidation and, therefore, markers of oxidative stress and can give an idea of the activity of antioxidant enzymes such as superoxide dismutase. However, these reactive oxygen metabolites are best detected in blood, urine or tissue samples (222,223). As we only analyse metabolites in faecal samples, we cannot see these oxygen-reactive metabolites. In addition, nitric oxide synthesis was identified as a functional module that decreased in our samples, which contradicts the decrease in superoxide dismutase, as this molecule reflects oxidative stress. Thus, with the study we have carried out, we cannot associate a reduction in the antioxidant enzyme in bacteria with a potential increase in oxidative stress in our samples, and we cannot determine whether bacteria are associated with more or less oxidative stress in our sample.

In addition, LC-MS cannot detect specific metabolites described as very important for tissue homeostasis, such as saturated fatty acids. Saturated fatty acids can originate from the host but also from the microbiota. In the multiscale study and completeness analysis 1, we identified an increase in butyrate synthesis and functional modules of butyrate production, although we identified a decrease in acetate synthesis and degradation. According to these results, we should have detected an increase in butyrate, but this seems less clear from this study alone concerning acetate. As saturated fatty acids are detected by gas chromatography and mass spectrometry, we have no idea of the quantity of saturated fatty acids in our sample, and we cannot conclude on their evolution in our sample and associate it with the activity of the microbiota and therefore with the role they could play in radiation-induced lesions.

Finally, with the proton ion, it is difficult to sequence components of the intestinal microbiota other than bacteria, even with shotgun analysis. We, therefore, only have a reflection of the activity of the microbiota through the spectrum of bacteria. Still, other communities of microorganisms could impact its functionality and, therefore, the host-microbiota relationship (224,225).

In conclusion, we have identified the arginine-proline pathway as an important element in our animals' radiation-induced lesion present at J56. It is a way to understand better the state of the symbiosis between host and microbiota. Still, it is essential to put into perspective that other factors could be involved in radiotoxicity but that we were unable to detect with the techniques used.

We performed correlation studies on each species detected in our samples, each functional It could mean the degraded host state is not severe enough to affect the microbiota. If the mutualistic symbiosis is not broken down, the microbiota could participate in the full recovery of the host in the longer term.

As regards the consequences of exposure to 3x14Gy, it appears to cause a breakdown in the mutualistic symbiosis between the host and the microbiota. This study highlighted periods when it would be interesting to treat to modulate the intestinal microbiota, for example, between D3 and D30 after the third dose of irradiation, where certain species and functions were positively correlated with the size of the ulceration at D56 to reduce the risk of developing a lesion, or after D40 after the third dose of irradiation to stimulate certain species involved in the production of proline in particular, and those involved in the degradation of glutamate, which could lead to better tissue repair. Unfortunately, we did not have this result before testing FMT in our experimental model.

III. Does FMT seem to be interesting in the context of PRD?

This project aims to study the modulation of gut microbiome and its interest in the context of PRD. While several ways of modulating gut microbiota exist, we have focused on FMT and we will explain why.

Several projects have focused on the effect of probiotics, prebiotic or even diet in the context of radiation-induced lesions and have obtained encouraging experimental results, so why consider in our project FMT?

Probiotics seem to be an encouraging treatment but have certain limitations. There are two ways of looking at probiotics: as a drug, with the idea of providing, not only a strain, but also

Why FMT

A. Probiotics all the molecules it produces (such as beneficial metabolites) and as a new element of the intestinal microbiota that would provide the missing element before its implantation. In the first case, implantation is not necessary, but the administration protocol may be difficult to implement. Little is known about how much to take and for how long por each probiotic. In the context of chronic diseases, if one considers the probiotic as a medication, we can think that it should be taken as such, i.e. every day. Regarding the dose, we have to make sure that there is a sufficient quantity of bacteria provided to ensure correct amount of therapeutical molecules produced. If we consider a species that must be integrated into the intestinal microbiota, we therefore need to implant the strain. But recently established ecological discoveries seem to converge on the idea that some species are highly interdependent on others to colonise an environment (106). With this concept, one needs to ensure that the gut microbiota into which the probiotic arrive contains species on which the probiotic strain is dependent which may appear complicated. It would also be necessary to ensure that the strain produces what it was intended for. Microorganisms can have great phenotypic plasticity when they change environment. New pathways can be activated and others inhibited, which can lead them to produce or consume elements different from the environment in which they were studied (226). It also appears that this phenotypic plasticity is not only dependent on environmental conditions but also on the social environment, i.e. the living beings that surround them (168).

The environment selects the phenotype, and if the behaviour of the probiotic strain in the environment where it arrives is not known, there is no guarantee that it will behave in the same way as in the environment from which it originates. Even if some species do not have a great capacity for plasticity, they remain sensitive to mutations and horizontal gene transfers that can modify their phenotype. In this context and compared to probiotics the strong advantage of FMT is that it provides a complete microbiota which might be thought to undergo less modification (but still) in terms of environmental change, only one component is modified namely the habitat, compared to a species alone or accompanied by others. In addition, it should have a better chance of establishing itself, competing and replacing another microbiota, especially if the latter has been disturbed. Because in the context of PRD the gut microbiome appears to be altered, it seems interesting to bring another healthy microbiota which is healthy to see how it could have beneficial effects to the global gut ecosystem.

Other treatment methods could be considered, with a different strategy that should lead to the same expected effect. With prebiotics and diets, the action on the microbiota is indirect. The principle is to create an environment favourable to the growth of interesting species that should be beneficial to the host. Prebiotics are to probiotics what diets are to FMT. Prebiotics and probiotics can temporarily modify the parameters of the host (prebiotics) and the microbiota (probiotics). While diets and FMT could completely modify by their use the physiology of the host (diet) and the microbiota (FMT). The environment will select the desired strains to be more abundant at the expense of others. Prebiotics seem to be an excellent way, in an environment undergoing small recent changes, to help restore the ecosystem more quickly than it would naturally. For example, when there is a change in diet or an infection with a pathogen, the organism could eject on its own. Diet seems to be the most radical and, therefore, the most effective in a stable host detrimental ecosystem. Acting on the environment seems an excellent solution but can also be difficult to implement. The choice of diet does not seem to be a limitation in regard to FMT as it is possible to think about the choice of donor for the transfer to ensure the best consortium of microorganisms for the donor. The main issue is the shift in habit required by a change in diet, not only in terms of cravings (food activates reward system (227) but also of the whole food-related organisation of the patient. Socially, it is also important to consider that patients might not have access to healthy and diverse food. Moreover, a change in diet can lead to an increase in food preparation time, especially if the patient lives in an environment where the people around him/her have a different diet. Furthermore, the increase in the consumption of prepared foods is a marker that people do not take an important capital in diet and/or do not have other choices (simpler and cheaper). A whole range of education about nutrition and consideration of the individual situation is needed.

As far as patient monitoring is concerned, even if it is counter-intuitive, it seems more difficult to change drastically a diet than use "drug". The advantage of medication is that the patient needs a prescription and delivery in a pharmacy. Therefore, the medical doctor and pharmacy staff can keep track of the last date of the prescription and the last dispensing. The idea is that it is easier to follow up and better ensure that the patient follows the treatment with medication than with diet. Despite this, the dietary change appears to be an effective way to achieve remission or reduce pathological features in some patients (228). However, further studies are needed on the effect of diet on certain diseases such as IBD, as the 2019 Cochrane review

B. Prebiotic and diet

seems to consider that the evidence is too weak to state with certainty that diet might be effective on this disease (229). In the context of PRD, it is possible to imagine a treatment that would prevent the gut microbiota from changing before the onset of symptoms, or even, for patients already suffering from PRD, to import a new microbiota that could weaken inflammation and help repair tissues, and thus relieve the patient's symptoms without them being a burden.

Compared to diet-related strategy from a care organisation point of view, FMT is a medical act and therefore subject to the constraints and duties related to standardisation and patient followup. FMT has not been experimentally studied with a localised irradiation protocol. It is for these reasons that we decided to work on FMT, for which the potential has not yet been exploited experimentally in the context of PRD and because it seems simpler with equivalent potential benefits.

From the results we have described and discussed above, we have concluded that FMT is not effective when administered to prevent radiation-induced toxicity in the rat colon but may be effective when administered as part of a curative protocol. However, before generalising this conclusion, some points for improvement of our protocol should be discussed.

As mentioned earlier, the gut microbiota is populated by a consortium of microorganisms, some of which are strict anaerobes and others in the colon. Because of their importance for the health of the host, it is essential to protect them when performing FMT. In the laboratory we do not have the equipment to work under anaerobic conditions. Therefore, we prepare our samples in ambient air, which could damage some species of the microbiota. This was confirmed in a recent study highlighting the importance of preparing samples under anaerobic conditions (230). The researchers prepared the samples under different conditions, anaerobic and aerobic.

They also studied the effect of freezing/thawing on samples prepared under anaerobic conditions. They found that bacterial viability (measured using 16S rRNA) decreased by 50% when the sample was prepared under anaerobic conditions, by less than 20% when prepared 2. FMT for preclinical PRD : element to consider for further experiment A. Preparation of the sample under aerobic conditions and by about 30% when prepared under anaerobic conditions and frozen and thawed. Regarding composition, they showed that samples prepared under aerobic conditions had fewer taxa than samples prepared under anaerobic conditions. Furthermore, they observed that samples prepared under aerobic conditions had lower butyrate production than samples prepared under anaerobic conditions. In conclusion, sample preparation is a crucial step, and probably, in my work, we did not have optimal preparation. Even if we transfer dead bacteria that might have an effect by stimulating the growth of some interesting species, it seems better to have as little loss as possible. If we administer a faecal microbiota with many dead bacteria, we can question its ability to modify the composition of the one living in the colonic ecosystem. In future FMT experiments, it would be essential to prepare the samples in the best condition to increase the probability of success.

As explained in the previous paragraph, our preparation conditions were not optimal and under these conditions, one could expect not to have many living microorganisms, and therefore a limited effect. However, we observed a positive effect on one experiment which could suggest that the gut microbiota does not need to be alive to produce an effect or that the effect comes from other elements of the transfer that are not microorganisms. The pads used to filter FMT have a millimetre-sized diameter. For example, the filters used to filter FMT have a millimetresized diameter. Therefore, microorganisms can pass as well as host components such as cells, vesicles or other molecules. When we measured the microbial community in the faecal samples, we found that some samples contained a large amount of host DNA, which implies that it is possible that some of the DNA in the sample comes from the host and not from the microbiota (in some cases the sample contained up to 30% host DNA) (Figure .44). This filtration method is expected to pass more components than the FMT supernatant, which is centrifuged before filtering, which may remove many molecules of interest, and therefore cannot claim that, compared to an FMT preparation, only the microbiota has been removed.

The freezing process could also change all the other molecules present in the transfer, but this seems unknown. Thus, in an FMT, we are not only introducing microbiota but also a whole ecosystem. Therefore, it may be worthwhile to carry out tests to identify what makes up an FMT and, if it is effective, which component, if not all, is most effective. What we could have done and didn't do, even in the samples where FMT didn't prove to be effective, which could have guided us in this reflection, is to measure the amount of 16S after doing the transfer, which

B. Elements constituting FMT

could allow us to see if some of our FMT has managed to take hold in the colonic environment.

This could be an interesting thing to do in the future.

In this project we focus on the effect that FMT could have on radiation-induced lesions in the rat colon, but it is possible to consider FMT as another type of treatment, for example, as a booster for certain treatments in the context of cancer therapy.

In recent decades, immunotherapy has gained ground in the context of cancer treatment.

Because immunotherapy works as a booster of certain elements of the immune system to have

Other use of FMT

A. Anticancer treatment efficiency an effect on the tumour, and because the microbiota is finely linked with the immune system, some researchers have become interested in the subject of microbiota in the context of anticancer treatments with immunotherapy. Patients who have cancer and are undergoing treatment are often prescribed antibiotics in the context of the development of bacterial infections. Thus, it has been possible for some researchers to carry out clinical studies on the effectiveness of certain immunotherapy treatments in patients who have received antibiotics compared to those who have not. Notably, a 2017 study showed that in a cohort of patients treated with an anti-PD1/PD-L1 mAb for non-small cell lung cancer, renal cell carcinoma or urothelial carcinoma, those who received antibiotics (before or during their treatment) had a lower progression free survival and overall survival than patients who did not receive antibiotics In addition, the authors found a correlation between the response to immunotherapy treatment and the presence of Akkermansia muciniphila, patient who better respond to their treatment have higher abundance of this bacteria in their microbiota (231). The idea that the microbiota can predict response or not to treatment has attracted much interest and various studies have looked at the role of the microbiota in patients who respond to cancer treatments other than immunotherapy (232). In the context of chemoradiotherapy for pelvic cancer researchers have highlighted that microbiota could boost the chemoradiation treatment. They showed that patients treated for cervical cancer with a higher pre-treatment Shannon score had better recurrence-free survival and better overall survival, as shown by the Kaplan-Meir curve. Although microbiota diversity did not distinguish patients by tumour characteristics, it did distinguish those who would respond positively to treatment from those who would not (233). The authors also found a correlation between a high Shannon index diversity and a high proportion of T cells and so connected gut microbiota with immunity.

There do not appear to be any clinical studies on the efficacy of radiotherapy alone about the composition of the intestinal microbiota in the context of antitumour treatment. However, experimental studies have looked into this question. In the context of a breast cancer model in syngeneic mice, Shiao et al. showed that the antitumour benefits of radiotherapy decreased when antibiotics were administered to the mice and increased when antifungal agents were administered to the mice. The authors attributed the reduced efficacy of radiotherapy treatment to the microbial composition, in particular to the increased abundance of fungal composition, and more specifically to Candida albicans, which, when administered after antibiotic treatment in the same mouse model, reduced the efficacy of radiotherapy in terms of its antitumour effect.

In contrast, in animals receiving Candida albicans, the addition of fluconazole, which targets this fungus species, increased radiotherapy's efficacy. In addition, the use of a KO mouse model for Dectinc-1, which is a receptor for fungal components (b-glycan) and the application of radiotherapy in this breast cancer model increased the antitumour effect of radiotherapy (234).

The composition of the microbiota, therefore, appears to play a role in the efficacy of radiotherapy. In conclusion, the gut microbiota appears to be closely linked to the success or failure of cancer treatments. Since the microbiota appears to be involved in the success or failure of cancer treatments, it is conceivable that it may also play a role in the development of side effects induced by these treatments.

Two clinical studies examined the microbial composition of patients treated with ipilimumab for metastatic melanoma (235,236). Both studies found that patients resistant to treatmentrelated colitis had a microbial composition enriched by the phylum Bacteroidetes. Both studies also addressed the inflammatory profile of patients but did not reach the same conclusion (Chaput et al., seemed to state that the inflammatory profile was higher in patients with a majority of Bacteroidetes, while Dubin et al., seemed to associate this microbial profile with less inflammation). As the parameters measured to judge the inflammatory profile are not the same in these two studies, it is difficult to distinguish between them on this point. However, Chaput et al., looked also at therapeutic efficacy and showed that the patients with the best response to the treatment were those who developed treatment-related colitis and that these patients had a microbial signature enhanced by Firmicutes (236). While the side effects of immunotherapy may be such that the patient decides to stop the treatment, it is very interesting to identify what might be correlated with these side effects in order to play on them to avoid them. However, the difficulty in cancer treatment is to maintain a favourable benefit/risk ratio and therefore to ensure that, for example, modulation of the microbiota to avoid the side effects of a treatment does not result in therapeutic failure. As these clinical studies do not involve large numbers of patients, it would be interesting to continue research on this topic to try to better identify measures to improve patients' response to treatment while reducing their risk of developing side effects. In the context of radiation-induced toxicity, researchers have been interested in the microbiota (169). Guo et al., exposed mice to whole-body irradiation and showed that the mice that survived best had a specific microbial signature. To confirm the radioprotective potential of this microbial signature, authors performed an FMT with faeces

B. Avoiding adverse events associated with anticancer therapies

Radiotherapy is an essential part of the treatment strategy for cancer patients. Thus, many people benefit from it. Despite all the efforts made in recent years to improve the risk-benefit ratio, exposure to ionising radiation can lead to side effects. In particular, in pelvic cancers, 90% of patients who receive radiotherapy develop acute toxicity from the beginning of the radiotherapy and up to three months after its end and 5-20% of patients develop late complications within 5 to 20 years after the radiotherapy. These complications were combined into a single condition by Andreyev and his team in 2010 and are now known as Pelvic

Radiation Disease. This condition can affect several organs, particularly the digestive system such as the colon and rectum. In a context where patients do not have access to treatments that allow them to recover from this pathology and where it can significantly deteriorate the lives of patients, it is crucial to continue research on PRD in order to understand better the pathophysiology and mechanisms that can be used to prevent and treat it in people already affected.

It is in this context that this thesis project is introduced. This thesis has three objectives; the first was to develop experimental models of PRD using fractionated irradiation protocols in order to get closer to what is done in clinical radiotherapy. This first objective allowed us to develop two models, one model of moderate and one model of severe radiation-induced colonic injury. The interest in the two doses allowed us to reproduce the least severe and most severe patterns of colonic epithelial damage that patients with PRD may present. The second objective was to measure the microbiota and the faecal metabolome over time before and after irradiation in order to identify whether the gut microbiota could play a role in the development of this radiation-induced colonic toxicity, as well as to identify the means by which the latter could be involved. This objective allowed us to better understand the dynamics of the colonic ecosystem by cross-checking its analyses with those measured in the host during the first objective. The third objective was to test the interest that TMF could have in the context of colonic radiotoxicity, in preventive or curative treatment.

The results obtained during the thesis allowed us to identify a dose effect on the host and on the microbiota. The model with the lowest lesions showed no difference in microbial composition and metabolome compared to the control. However, exposure to ionising radiation affected the tissue with the appearance of lesions and an immune recruitment marked by an increase in macrophages and mast cells in the longer term. In the final analysis, some animals showed no lesions compared with the control, which allowed us to deduce that the 3x10Gy model could be regenerative. However, for those animals that still showed lesions at the last analysis, we cannot confirm that they would have fully recovered by shifting the sacrifice time to later. The moderate model seems to be at the interlude between optimal repair and perpetuation of injury to an unknown time. In the analysis of the severe model, we could see that the microbiota and metabolome showed changes over time compared to the controls. Thus, in this model, in view of the damage found and the alterations in the microbiota, the hostmicrobiota symbiosis seems to have changed from mutualistic to parasitic. Through mathematical and statistical analysis we were able to identify that bacteria were involved in the size of the lesion in the later stages of this study. However, we also identified several species negatively correlated with lesion severity in the last time of analysis. Regarding the functional modules associated with the bacteria and the metabolome, some follow a similar dynamic as some bacteria. The arginine-proline pathway is among these metabolite pathways with the same dynamics as microbial species. According to our results, the increase in proline seems to prevent the development of ulceration, and the rise in glutamate over time appears to be involved in the lesion development. Stimulating proline biosynthesis and reducing glutamate biosynthesis would therefore reduce radiation-induced lesions. It would be interesting to modify the microbiota before irradiation to see if lowering radiation-induced lesions in the colon is possible. If this is the case, and even if not, the involvement of proline and glutamate could be studied to see if our hypothesis can be validated.

Finally, regarding the results of FMT, we have observed that it does not seem effective in our moderate and severe models in a preventive context. However, the protocols developed to perform this FMT could be modified to be more optimal. Only after improving these protocols will we be able to confirm that FMT cannot be of interest in a preventive context. Moreover, FMT allowed the animals to present less severe lesions in a curative context for the moderate model, with a start of treatment a few days after the last exposure to ionising radiation.

Thus, the breakdown of the mutualistic symbiosis between host microbiota appears to be impaired after exposure to high doses of radiation. As a preventive measure, FMT did not improve the condition of animals with the most severe lesions. After improving the protocol, it would be interesting to re-test its use in preventive and curative treatment to see if it can restore a mutualistic relationship between the host and the microbiota. However, if the ineffectiveness of FMT is confirmed in the experimental model of PRD, the use of diet could be another solution for modifying the intestinal microbiota and preventing or helping to repair radiation-induced lesions.

Introduction

Cancer is the second leading cause of death globally. Radiation therapy, an established part of treatment of both primary and recurrent pelvic malignancies, is used in almost 50% of cases and concerns millions of people worldwide each year. Improvement in treatment in the last decades has led to a threefold increase in the number of cancer survivors over the last 30 y. The efficacy of radiation therapy requires an optimal compromise between tumor control and healthy, normal tissue tolerance; this is referred to as the benefit/risk ratio. Following radiation therapy, healthy tissue around the tumor can be damaged, initiating in 90% of patients acute, and in 5% to 10% of patients, chronic side effects. A high incidence of acute symptoms is reported during or shortly after the end of the radiation therapy. In some cases, depending on dose, volume, as well as individual susceptibilities, severe acute symptoms may result in treatment suspension, and thus failure of tumor control. Moreover, more than 10 y after the end of the treatment, patients may exhibit persistent overlapping symptoms (diarrhea, constipation, bloating, pain), resulting from multiple visceral organ dysfunctions. All these symptoms were recognized in 2010 as a new disease entity called "Pelvic Radiation Disease" (PRD). 1 Digestive mucosa constitutes a physical barrier, the first line of defense against pathogen infiltration in the organism. Due to the high proliferative capacity of crypt epithelial stem/progenitor cells, colonic mucosa is highly radiosensitive. After local radiation exposure, a complex cascade of biological effects, i.e. from damage of multiple biomolecules to cell phenotype modification and/or death, may induce disruption of barrier integrity. Colonic barrier disruption may drive bacterial translocation and likely the maintenance and/or exacerbation of mucosal inflammation. 2 After local radiation exposure, in the absence of acute immune response control, chronic inflammation could lead to persistent and irreversible damage. So, protecting the colonic mucosa from radiation toxicity and therefore from the progression of inflammation might result in the alleviation of acute and chronic side effects and should improve the patient's quality of life and/or reduce the risk of life-threatening conditions.

In 2014, Andreyev suggested that intestinal microbiota could be implicated in the initiation and/or maintenance of radiation-induced intestinal toxicity. 3 In patients receiving pelvic radiation therapy, there is a loss of microbial diversity, which is more pronounced in those with side effects like diarrhea. Among observed abundance variation, bacteria belonging to the genus Faecalibacterium were reduced. 4,5 Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant anaerobic bacteria in the human colon (up to 5%) and so far the only member of the Faecalibacterium genus. Preclinical experiments performed in animal models of inflammatory bowel disease have previously demonstrated the therapeutic benefit of prophylactic treatment with F. prausnitzii, which reduces colonic damage and leads to increased survival of animals. 6-10 Earlier studies demonstrated an ability of F. prausnitzii to reduce histological and functional colonic barrier impairment. The beneficial effects of F. prausnitzii were associated with its tendency to counterbalance dysbiosis and with local or systemic anti-inflammatory effects. 6,7,9,11,12 Sokol et al. 6 reported that live F. prausnitzii administration prevents an acute TNBS-induced increase in the proinflammatory cytokine IL-12, but only tends to increase the anti-inflammatory cytokine IL-10 in the colon. Some other pro-inflammatory cytokines are also reduced by live F. prausnitzii administration in the colon of mice with chronic low-grade inflammation: TNF-α, IFN-γ and IL-6. 9,10 While these studies have outlined a protective effect of F. prausnitzii treatment on the colonic mucosa, probably in part via management of the inflammatory response, the overall effect of F. prausnitzii on the proliferation and self-renewal of crypt epithelial cells remains unknown.

In this study, we made the assumption that F. prausnitzii administration might preserve colonic mucosa from radiation damage by controlling the immune response. Our objectives were to test the effectiveness of prophylactic F. prausnitzii treatment on radiation-induced colonic mucosal barrier breakdown. We analyzed various parameters to assess the maintenance and/or disruption of colonic mucosal barrier integrity, such as the histological structure and self-renewal of the colonic epithelium, paraand trans-cellular permeability, microbiota composition/diversity and the inflammatory process. In a context of radiation-induced colonic mucosal toxicity and dysfunction, we found that prophylactic F. prausnitzii treatment had protective effects. We demonstrated that this effect is associated with the ability of F. prausnitzii to prevent acute para-cellular permeability and therefore the nonspecific immune response. Our results also suggested for the first time that the therapeutic benefit of F. prausnitzii might also follow activation of protected epithelial cells and underlined the probable involvement of differentiated epithelial cells, principally tuft cells.

Materials and methods

housed in standard conditions for a minimum of 1 week before experimentation. They were allowed free access to water and fed standard pellets.

Bacterial strains and growth conditions

Faecalibacterium prausnitzii strain A2-165 (DSMZ collection, Braunschweig, Germany) (DSM No 17677) was grown in LYBHI medium (brain-heart infusion medium supplemented with 0.5% yeast extract) (Difco, Detroit, USA) supplemented with 1 mg/mL cellobiose (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), 1 mg/mL maltose (Sigma-Aldrich), and 0.5 mg/mL cysteine (Sigma-Aldrich) at 37°C in an anaerobic chamber (90% N 2 , 5% CO, 2 and 5% H 2 ). Bacteria were harvested by centrifugation, washed in PBS, re-suspended in PBS containing 20% glycerold stored at -80°C.

Irradiation protocol

Rats were anesthetized by isoflurane inhalation and a single 29 Gray (Gy) dose of irradiation was delivered by a medical accelerator (Alphée) through a 2x3cm window centered on the colorectal region. Alphée is an accelerator-type radiation source (maximal energy is 4 MeV with an average energy of about 1.5 MeV; 30 kA).

Experimental design

Two groups of rats were submitted to colorectal irradiation at a single 29 Gy dose and one group was not irradiated (control group). The irradiated rats received prophylactic treatment with F prausnizii or received only vehicle. Nonirradiated group received vehicle. Three days before irradiation, the day of irradiation, and 2 d after irradiation, 1.10 9 CFU of bacteria or PBS was intragastrically administered once daily. Rats were sacrificed, 6 h, 3 d after irradiation. The study groups were as follows: Control group (Ctrl PBS): nonirradiated and PBS-treated rats, Irradiated group (IRR PBS): irradiated and PBS-treated rats, and -F. prausnitzii treated group (IRR Fprau): irradiated and F. prausnitzii -treated rats.

In the first batch of the experiment, the colonic barrier integrity was analyzed on colonic sections by histological analysis, immunohistochemistry/ immunofluorescence staining and TUNEL assay, 6 h or 3 d after irradiation. One independent experiment for each time of sacrifice (N = 1, 5 animals per groups) was performed (8 to 20 independent colonic sections per rats were analyzed) and structural damages (morphological crypt atypia or crypt loss; HES staining), epithelium renewal ability (TUNEL and PCNA-positive crypt epithelial cells in apoptosis or proliferation, respectively, SOX9-positive progenitor/stem cells and DclK1positive Tuft cells) and inflammatory process (mucosal infiltration of MPO-positive neutrophils and CD68-positive macrophages and IL-18positive crypt epithelial cells) were assessed.

In a second batch of the experiment, the colonic barrier integrity was analyzed by measuring ex vivo colonic trans-and para-cellular colonic permeability. Three independent experiments were performed (N = 3, between 8 and 12 animals per group for each experiment).

In a third batch of the experiment, 3 d after irradiation, fecal microbiota composition by the V3-V4 hyper-variable region of the 16SrRNA sequencing and also mucosal cytokine concentration using ELISA bio-plex cytokine assay (23 cytokines including IL-18) were assessed. One independent experiment was performed (N = 1, between 9 and 12 animals per group).

In a fourth batch of the experiment, the colonic barrier integrity was analyzed 7 d after irradiation by histological analysis performed on colonic sections stained with HES (See Supplementary materials and methods). One independent experiment was performed (N = 1, 8 animals per groups, 20 independent colonic sections per rats were analyzed) and the severity of colonic mucosal ulceration was measured.

Histological and damage analysis

Animals were sacrificed by isoflurane inhalation 3 d after colorectal irradiation. Distal colons were removed, fixed in 4% formaldehyde, cross-sectioned in four equal pieces and embedded in paraffin. Paraffin-embedded colons were cut on a rotary microtome (Leica Microsystems AG, Wetzlar, Germany) into serial circular sections of 5 µm, in 5 series spaced by 250 µm and stained with hematoxylin-eosinsaffron (HES). Each slide was composed of four circular sections of the cross-sectioned colon representative of lesion along the colon. The severity of histological damage of the mucosa on each colonic circular section was assessed using a lesion score with the Histolab software (Microvision Instruments, Lisses, France). Determination of the injury score was based on (1) inflammatory infiltration (modest inflammatory infiltration at the base of the crypt leading to slight crypt detachment from the lamina propria; 0< score<1, or inflammatory infiltration in all the lamina propria; 1< score<2), (2) epithelial damage (crypt morphology atypia 0< score<1, and loss of epithelial crypts (1< score<2) or both (1 + 2; in this case graduation of the injury was: score 0 = null; 0< score<1 = slight; 1< score<2 = moderate and score>2 = severe). The injury score was calculated for each animal group. Morphometric analysis of crypt depth was also performed on HES-stained slides.

Immunohistochemistry and immunofluorescence

For immunohistochemistry, sections were deparaffinized and hydrated. Tissue sections were treated with 0.1% Triton X-100 (Sigma-Aldrich) in PBS 1x (Life Technologies) at room temperature (RT) for 10 min. Then, endogenous peroxidases were inhibited by incubation with 3% H 2 O 2 in methanol at RT for 10 min. Citrate buffer (10 mM, pH6) (Zytomed) was used as an antigen retrieval solution; incubation lasted 15 min at 350 W. After saturation (X0909, Dako), rabbit anti-SOX9 (ab185230, Abcam, 0.803-0.971 mg/mL, for stem/progenitor epithelial cell staining) diluted to 1/2000 and rabbit anti-MPO (IMG-803971, Abcam, for neutrophil staining) diluted to 1/75 was applied for 16 h at 4° C. After saturation (X0909, Dako), mouse anti-PCNA antibody diluted to 1/10000 (M0879 Dako, 525 µg/mL, for proliferating cell nuclear staining), and mouse anti-CD68 (MCA341R, AbDserotec, for resident macrophage staining) diluted to 1/200 was applied for 1 h at 37°C. Sections were then incubated for 30 min at RT with, respectively, antirabbit-HRP Ig (D13-110, GBI Labs) or anti-mouse-HRP Ig (D55-18, GBI Labs). Staining was developed with HRP Green (ZUC 070-100, Zytomed). The sections were then counterstained with nuclear fast red (H3403, VectorLabs), dehydrated, and mounted. The negative control was assessed with rabbit and mouse IgG (Dako, X0931) instead of the analyzed primary antibodies.

For immunofluorescence, sections were deparaffinized and hydrated, and incubated with 3% H 2 O 2 in methanol followed by an incubation in citrate buffer. After saturation (X0909, Dako), rabbit anti-Dclk1 (ab109029, Abcam, for tuft cell staining) diluted to 1/300 or rabbit IL-25 (IL-17E, ab108530, Abcam) diluted to 1/500 was applied for 16 h at 4°C. After saturation (X0909, Dako), rabbit anti-IL-18 (HPA003980, Atlas antibodies) diluted to 1/50 was applied for 1 h at 37°C. For DclK1 or IL-18, sections were then incubated with donkey anti-rabbit Alexa Fluor 488 (A21206, Invitrogen) diluted to 1/200 for 1 h at RT. For IL-25, sections were then incubated with goat antirabbit Alexa Fluor 568 (A11011, life technologies) diluted to 1/200 for 1 h at RT. For Dclk1/IL25 double staining, immunostaining of IL25 and DclK1 were performed one by one starting by IL25. Slides were counterstained with DAPI and mounted with Vectashield (H-1000, Vector). The negative control was assessed with rabbit IgG (Dako, X0903) instead of the analyzed primary antibodies.

Ex vivo para-and trans-cellular permeability measurement in using chambers

Three days after colorectal irradiation rats were sacrificed. Immediately after sacrifice, distal colons were removed, cut along the mesenteric border and mounted in Ussing chambers (Corning Costar Corporation, Harvard Apparatus, France) with a flux area of 1.78 cm2. Colonic cellular permeability to small and large molecules was measured through the mucosal-to-serosal passage of fluoroisothiocyanate (FITC)-Dextran 4 kDa (FD4; Sigma-Aldrich, Saint Quentin Fallavier, France) and intact horseradish peroxidase 44 kDa (HRP; Sigma-Aldrich, Saint Quentin Fallavier, France) respectively, added simultaneously in the mucosal compartment. After 20 min of equilibration, 600 µL of buffer solution on the mucosal side was replaced by 300 µL of FD4 (2.2 mg/mL) and 300 µL of HRP (0,4 mg/ml). After 1-h incubation, colonic permeability to FD4 was determined by measuring the fluorescence intensity at 485 nm/525 nm using an automatic Infinite M200 microplate reader (Tecan, Lyon, France). Cellular permeability to intact HRP was determined by enzymatic assay 13 for specific e1812867-4 HRP activity found in the serosal and mucosal compartment using a microplate reader (Tecan). Permeability was calculated as the ratio of flux/ concentration, as previously described 14 and expressed in cm/second. Finally, comparison between groups was assessed by calculating relative percentage according to Ctrl PBS permeability.

Colonic protein extraction and Bio-Plex cytokine assay

Colon mucosa proteins were extracted following the instructions for the Bio-Plex Cell Lysis kit (171304011, Bio-Rad). Colonic concentrations of 23 cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, IL-18, IFN-γ, TNF-α, VEGF, G-CSF, M-CSF, GM-CSF, GRO/KC, MCP-1, MIP-3α, MIP-1α and RANTES) were measured in extracts from colon tissue using the Bio-Plex Pro Rat Cytokine 23 (12005641, Bio-Rad) according to the manufacturer's instructions. This Bio-Plex 200 system uses fluorescently dyed beads to detect different types of molecules in a single well of a 96-microwell plate, requiring low sample volumes. Briefly, premixed beads coated with target antibodies (50 µL) were added to each well and washed twice with Bio-Plex wash buffer. Standards (eight concentrations ranging from 32000 to 1.95 pg/mL) and samples (50 µL) were then added to the wells, followed by shaking at 1100 rpm for 30 sec and incubation for 30 min with shaking at 300 rpm at RT. Wells were then washed 3 times with Bio-Plex wash buffer, and premixed detection antibody (25 µL) was added to the wells. This was followed by shaking at 1100 rpm for 30 sec and incubation for 30 min with shaking at 300 rpm at RT. Wells were again washed 3 times with Bio-Plex wash buffer and streptavidin-PE (25 µL) was added to the wells. Incubation was for 10 min with shaking at 300 rpm. Wells were washed 3 times with Bio-Plex wash buffer and the beads were resuspended in 125 µL of Bio-Plex assay buffer. With this technology, relevant inflammatory cytokines could be detected in a single run. Dyed beads were read on the Bio-Plex analyzer. One laser classified the beads and determines the cytokine being detected, and a second laser determined the magnitude of the phycoerythrin-derived signal, which is in direct proportion to the amount of molecule bound. Cytokine concentrations were derived by interpolating the measured fluorescence intensities to standard curves and by correcting using the corresponding dilution factor employed to achieve the minimum volume for analysis. Bio-Plex Manager software (Bio-Rad) was employed to calculate cytokine concentrations. To avoid interassay variations, all samples were analyzed with the same kit on the same day.

Fecal microbiota analysis

Total bacterial DNA was extracted from the fecal samples according to the protocol described in Godon et al. 15 DNA concentration, purity, and integrity were determined using a NanoDrop instrument. The size distribution of the extracted DNA estimated by 1% agarose gel electrophoresis showed that most of the DNA was high molecular weight (>20 kb) with no significant shearing. These observations suggest that the extracted DNA was of good quality and suitable for downstream processing.

The V3-V4 hyper-variable region of the 16S rRNA gene was amplified with the primers MSQ-16SV3F (CTTTCCCTACACGACGCTCTTCCGATCTACG-GRAGGCWGCAG) and MSQ-16SV4R (GGAGTTCAGACGTGTGCTCTTCCGATCTTA-CCAGGGTATCTAATCCT). The PCR reactions were performed using 10 ng of extracted DNA, 0.5 μM primer, 0.2 mM dNTP, and 0.5 U of the DNA-free Taq-polymerase, MolTaq 16S DNA Polymerase (Molzym). The amplifications were carried out using the following profile: 1 cycle at 94°C for 60 s, followed by 30 cycles at 94°C for 60 s, 65°C for 60 s, 72°C for 60 s, and finishing with a step at 72°C for 10 min. The resulting PCR products were purified and sent to the @BRIDGe platform (INRA, Jouy-en-Josas) for sequencing using Illumina MiSeq technology.

Sequences were analyzed using the Galaxysupported pipeline FROGS to produce abundance tables of Operational Taxonomic Units (OTUs) and their taxonomic affiliation. The successive steps involved de-noising and clustering of the sequence into OTUs using SWARM; chimera removal using VSEARCH; and taxonomic affiliation for each OTU using RDP Classifier on the SILVA SSU 123 database. Statistical analyses were performed using "R" language and environment version 3.2.3. α-and β-diversity measurements and analysis of the differences in OTUs between samples was performed using the add-on package "Phyloseq" 16

Statistics

All data are presented as means (SEM). Statistical analyses were performed using Graph Pad Prism 5.0 (GraphPad, San Diego, CA). Statistical significance was determined using one-way ANOVA followed by Tukey's posttest for multiple groups, or a t-test when changes were compared between two groups. Statistical significance was set at p <.05.

Differences between the cytokine concentrations were analyzed using generalized least squares (GLS) models with group as nominal covariate (Controls, irradiated and Irradiated+Fprau). The GLS function of the nlme R package offers a variety of variance structure specifications to handle violations of the variance homogeneity assumption in order to obtain correct standard errors and statistical significance. In each case, the selection of the optimal model was conducted using the likelihood ratio for nested models and the AIC otherwise. The significant level was fixed at p < .05 after Benjamini-Hochberg discovery rate (FDR) correction.

Results

Prophylactic F. prausnitzii treatment reduces radiation-induced histological damage to the colonic epithelium

The colonic epithelium forms a selective barrier against the penetration of luminal contents and then commensal and pathogen bacterial translocation. Physical barrier integrity of the colon was investigated in scoring structural damage of the colonic epithelium on histology slides (Figure 1). In the control group, the histological damage score of the colonic epithelium was 0.40 ± 0.23 coming from rare zone with inflammatory infiltration and slight crypt detachment from the muscularis mucosa. Three days after colorectal irradiation, the score reached 2.72 ± 0.06 (p < .0001 vsvsvs control rats) corresponding to an increase in the number of sections with crypt morphological atypia (disorganized and deformed colonic epithelial crypts) or crypt loss but also with inflammatory infiltration in all the lamina propria. Compared to rats subjected to irradiation alone, in those pretreated with F. prausnitzii we showed a significant reduction in the severity of the histological damage score (2.15 ± 0.09 in irradiated and F. prausnitzii treatment vs 2.71 ± 0.06 in irradiated rats, p = .0013). Histological images and the scoring of parameters individually (Figure 1(c)) show that an increased score in irradiated rats treated by F prausnitzii in comparison with control rats is mostly dependent on modest inflammatory infiltration leading to slight crypt detachment from muscularis mucosa but infrequently dependent of crypt morphological atypia. So, F prausnitzii seems to maintain normal crypt morphology. Our results demonstrate an ability of F. prausnitzii to partially protect the structural integrity of the colonic mucosa from colorectal irradiation and suggest it has the potential to preserve the epithelium as a physical barrier against bacterial translocation. To support this assumption the following experiments looked at how F. prausnitzii can regulate the function of the colonic epithelial barrier.

Prophylactic F. prausnitzii treatment prevents radiation-induced disruption of the function of the colonic epithelial barrier

Low trans-and/or para-cellular permeability is a primordial index of the functional integrity of the colonic barrier. In this study, ex vivo colonic permeability using Ussing Chamber systems to measure trans-cellular permeability to intact horseradish peroxidase (HRP, 44KDa) and paracellular permeability to fluorescein isothiocyanate-dextran 4 (FD4, 4KDa) were quantified. At 3 d, colorectal irradiation in rats pre-treated with F. prausnitzii did not change transcellular permeability to HRP (Figure 2(a)). At the same time after colorectal irradiation, we found a 2.3-fold increase in paracellular permeability to FD4 (Figure 2(b), p < .05), neutrophil infiltration, as demonstrated by the increased number of MPOpositive neutrophils and expression of GRO/KC (a specific neutrophil chemo-attractant) in the colonic mucosa (Figure 2(c)¸2(e)), p < .01, p < .05, respectively), and probably antimicrobial activity, as suggested by increased expression of macrophage inflammatory protein 3α(MIP3α) by mucosal cells (Figure 2(f)), p < .05). Altogether, these findings point to colonic barrier breakdown 3 d after colorectal irradiation. Pre-treatment with F. prausnitzii protected rats from acute radiation-induced paracellular hyperpermeability. Permeability values in irradiated rats pre-treated with F. prausnitzii remained close to basal level (Figure 2(b)). F. prausnitzii also prevented radiation-induced neutrophil infiltration, as shown by control levels of MPO-positive neutrophils and GRO/KC expression in the colonic mucosa after F. prausnitzii administration (Figure 2(c), 2(e)). MIP3α, remained elevated in irradiated rats pre-treated with F. prausnitzii (Figure 2(f)), p < .05).

Prophylactic F. prausnitzii treatment increases resident macrophages in the colonic mucosa of rats subject to colorectal irradiation

Pre-treatment of irradiated rats with F. prausnitzii significantly increased CD68-positive resident macrophages in the colonic mucosa by 47.60%, while in rats not pretreated there was only a trend of increased macrophages (Figure 2(d)). Resident macrophages of the colon do not proliferate and appear to lack receptor-mediated chemotactic activity. Recruitment of circulating precursor monocytes seems to be the major source of cells that increase the number of resident macrophages after F. prausnitzii treatment. The chemokine RANTES plays a role in immune cell recruitment as monocytes, mast cells and lymphocytes, but its expression was reduced in rats not pretreated and in rats pre-treated with F. prausnitzii compared to control rats (Figure 2(g)), p < .05, p < .01, respectively). Nevertheless, a borderline significant (p = .06) increase in MCP-1 in the colonic mucosa is suggestive of monocyte recruitment (Figure 2(h)). Our results also suggest that potential-recruited monocytes could be differentiating into macrophages, as the radiation-increased MIP-3αlevel in the colonic mucosa remained high in rats receiving F. prausnitzii treatment 17 (Figure 2(f)).

Colorectal irradiation with or without F. prausnitzii treatment doesn't modify gut microbiota

We investigated whether colorectal irradiation with or without pre-treatment by F. prausnitzii involved modifications of the gut microbiota by analyzing the intestinal bacterial composition of the different experimental groups (Figure 3). We found no differences between groups in α-diversity, which measures the taxonomic richness of the fecal microbiota communities (Figure 3(a)). In addition, β-diversity analysis, which measures the degree of similarity between the gut microbial communities, revealed no clustering of the rats according to the F. prausnitzii or PBS pretreatments before irradiation (Figure 3(b)). Bacteria composition analysis at the phyla level showed no differences between the groups (Figure 3(c)).

Prophylactic F. prausnitzii treatment increases the production of the cytokine IL-18 by crypt epithelial cells of irradiated rats

To study the acute and local inflammatory response prophylactic F. prausnitzii treatment, colorectal irradiation induced up-regulation of proinflammatory cytokines IL-1β and IL-18 by mucosal cells (Figure 4(a-b), irradiated rats vs irradiated and F. prausnitzii treated rats p < .05). As increased levels of IL-1β and IL-18 cytokines were not seen after irradiation alone (Figure 4(a-b)) or after bacteria administration alone (data not shown), it is likely that F. prausnitzii treatment and radiation exposure induced synergic effects on the production of these cytokines by colonic mucosal cells. In the colonic mucosa, IL-18 is expressed in a wide range of immune cells like macrophages as well as in epithelial cells. Immunohistochemistry on colonic tissue slides was performed to study the cellular origin of IL-18 production after F. prausnitzii treatment (Figure 4(c-d)). Even though we observed immunostaining of IL-18 in some immune cells localized in the lamina propria of the colonic mucosa (Figure 4(c), arrow), epithelial cells were the major source of its production. In control rats, we observed cytoplasmic immunostaining of IL-18 in the entire array of crypt epithelial cells. At 3 d, colorectal irradiation slightly reduced IL-18 production and immunostaining was more diffuse in the epithelial cytoplasm. In comparison with control rats, pre-treatment of irradiated rats with F. prausnitzii increased IL-18 production by epithelial cells, mostly those localized at the base of the crypt, suggesting an enhancement of basal production of IL-18 in the progenitor/stem cell compartment of the mucosa. We then analyzed whether increased IL-18 production by epithelial cells at the crypt base was associated with regulation of stem/ progenitor cells.

Prophylactic F. prausnitzii results in a protection of a pool of crypt epithelial progenitor/stem cells and differentiated epithelial tuft cells from colorectal irradiation

We assessed the number of epithelial stem/progenitor cells on colon histology slides stained by Sox-9 antibody (Figure 5(a), 5(e)). The number of Sox-9-positive stem/progenitor cells per crypt had decreased by 14.30%, 3 d after colorectal irradiation (28.93 ± 0.15 in control rats vs 24.79 ± 0.21 in irradiated rats, p < .001). In irradiated rats receiving prophylactic F. prausnitzii treatment, the number of Sox-9-positive cells stayed close to control level, with a slight but significant increase (29.87 ± 0.23 in irradiated and F. prausnitzii treatment vs 28.93 ± 0.15 in control rats, p < .01). This demonstrated that F. prausnitzii pre-treatment prevented radiation-induced loss of crypt epithelial stem/progenitor cells. We then investigated the ability of F. prausnitzii to preserve epithelial differentiated cells after colorectal irradiation. We focused on tuft cells, which are identified as an anatomically and functionally distinct epithelial cell entity. They are key regulatory intestinal niche cells and may well constitute one of the niche components. 18 We measured the number of tuft cells on colon histology slides stained by Dclk1 antibody (Figure 5(b), 5(f)). In the colonic mucosa of control rats, there were an estimated 303.90 ± 15.10 Dclk1-positive tuft cells per slide. Three d after colorectal irradiation, the number of Dclk1-positive cells was only 195.7 ± 18.88, corresponding to a decrease of 35.6% of tuft cells (p < .001), and remained at control level in rats pretreated with F. prausnitzii (264.90 ± 26.10). This shows that prophylactic F. prausnitzii treatment also results in a protection of colonic-differentiated Dclk1-positive tuft cells from colorectal irradiation. After luminal infection by helminth parasites, cytokine IL-25 secreted by tuft cells stimulates stem/progenitor cells to regulate epithelial remodeling. 19,20 We then performed double staining of colon histology slides by Dclk1 and IL-25 antibody (Figure 5(d), 5(g)). In control rats, we found that among 303.90 ± 67.53 Dclk1positive tuft cells per slide only 13.20 ± 3.65 produce cytokine IL-25. Three days after colorectal irradiation, co-staining by Dclk1 and IL-25 was observed in 1.87 ± 0.77 tuft cells per slide, corresponding to an 85.80% decrease compared to controls (p < .01). In irradiated rats pretreated with F. prausnitzii, the number of tuft cells co-stained by Dclk1 and IL-25 remained at basal level (10.41 ± 1.92). F. prausnitzii also preserved the basal level of differentiated tuft cells that could provide an optimal microenvironment for stem cell function.

Prophylactic F. prausnitzii treatment results in maintaining the self-renewal of colonic epithelium exposed to radiation and reduces mucosal ulceration

We were then interested by the renewal ability of colonic epithelium, enhancement, or maintenance of which in response to exogenous stressors is key to physical barrier integrity. We first studied whether F. prausnitzii-induced protection of crypt epithelial stem/progenitor cells and tuft cells in irradiated rats was also associated with the ability of crypt epithelial cells to proliferate. We analyzed the number of proliferating crypt epithelial cells on histology colon slides stained by PCNA antibody (Figure 6 F. prausnitzii pre-treatment to preserve the basal level of epithelial self-renewal even if rats are exposed to colorectal irradiation and support its acute effects on the preservation of colonic epithelial barrier integrity. The benefit provided by F. prausnitzii pre-treatment was sustained until 7 d after colorectal irradiation (Figure 6(d), Supplementary materials and methods). Prophylactic F. prausnitzii treatment reduced colorectal irradiation-induced mucosal ulceration by 52.80% at 7 d (4546.50 ± 650.08 µm in irradiated rats vs 2146.70 ± 458.50 µm in irradiated rats treated with F. prausnitzii; *p < .01).

Discussion

This study demonstrated that prophylactic treatment with F. prausnitzii induces an acute protection of the colonic mucosal barrier from radiation exposure and therefore maintains its functional integrity. The therapeutic benefit of F. prausnitzii after irradiation is provided by its ability to prevent the increase of paracellular permeability, to enhance epithelial self-renewal and to preserve differentiated epithelial cells. The maintenance of colonic barrier integrity by F. prausnitzii is associated with macrophage recruitment and cytokine production by mucosal cells, mostly epithelial cells, as shown specifically for IL-18. We also found that F. prausnitzii treatment results in the preservation of colonic Dclk1/IL25-positive tuft cells.

Colorectal irradiation led to enhancement of colonic paracellular permeability and to neutrophil infiltration in the mucosa, which are indexes of mucosal barrier breakdown. We also quantified in the colonic mucosa a significant increase of the chemokine GRO/ KC, a specific neutrophil chemoattractant, which is suggestive of a perpetuating cycle of neutrophil recruitment. We also showed in the mucosa a significant increase of macrophage inflammatory protein 3α (MIP3α), which is provided mostly by epithelial cells, but also by other cells such as innate and adaptative immune cells. 17,21,22 Given that MIP3α is co-expressed with its physiological receptor CCR6 in neutrophils and that CCR6 expression is increased in activated neutrophils, 23 the possibility of autocrine and/or paracrine signaling involving MIP3α, which increases neutrophil recruitment within colonic mucosa 23 can also not be excluded 3 d after colorectal irradiation. Moreover, by its antimicrobial activity, 24 increased secretion of MIP3α by other mucosal cells may, in synergy with neutrophil recruitment and activation, help protect against bacterial invasion. Three days after irradiation, we noted no modification of the production by mucosal cells of pro-inflammatory cytokines, and even quantified a significant decrease in the chemokine RANTES. Expression of RANTES is increased in activated epithelial cells, endothelial cells, fibroblasts, monocytes/macrophages within hours of stimulation 25 and is induced late (3-5 d) after T lymphocyte activation. 26 A colorectal irradiationinduced decrease in the expression of RANTES may be due to the radiosensitivity of many mucosal cell types leading to the deregulation of their molecular machinery and/or death and then to malfunction of the local immune response depending on monocyte/ macrophages and T lymphocyte activation/and/or mobilization. 27-31 Finally, under our experimental conditions, 3 d after colorectal irradiation seems to be too short a time to identify a shift in fecal microbial composition. Indeed, Gerassy-Vainberg et al. 32 showed that targeted rectal radiation-induced timedependent modification of fecal microbiota composition and that the radiation-induced unique microbial signature was most evident at 6 weeks. In conclusion, our results seem to be consistent with a nonspecific innate inflammatory response (neutrophil infiltration and activation) to colorectal irradiation at 3 d, with mostly phagocytic and antimicrobial effects.

In this context of radiation exposure, prophylactic treatment with F. prausnitzii had a protective effect on impairment of the colonic mucosal barrier. We first demonstrated that oral administration of F. prausnitzii prevented radiation-induced increase in acute (3 d) para-cellular permeability and in the nonspecific innate inflammatory response orchestrated by neutrophils after their mobilization (reduction of the number of neutrophils and GRO/KC expression in the colonic mucosa). The protective effect of F. prausnitzii on hyperpermeability has already been shown in a model of inflammatory bowel disease with chronic low-grade inflammation. 9,10 In rodents receiving F. prausnitzii treatment for management of acute colitis, histological examination of the colon showed, as we quantified in our study in the colonic mucosa, a relatively low level of neutrophil infiltration. 7 F. prausnitzii also reduced MPO activity in a model of inflammatory bowel disease with chronic mild/severe grade colitis, 33 thus highlighting the ability of F. prausnitzii to reduce neutrophil activation in this context. Results obtained by Sokol et al. demonstrated an ability of F. prausnitzii supernatant (SN) to reduce in vitro IL-1β-induced IL-8 secretion by colonic epithelial cells (caco-2 cells). 6 As IL-8 is known to be involved in neutrophil recruitment, the findings of Sokol et al. support our results and also suggest the involvement of epithelial activation in F. prausnitzii metabolite effects.

We then demonstrated that prophylactic treatment with F. prausnitzii also decreased epithelial damage at 3 d, leading to a reduced area of mucosal ulceration at 7 d. In our experimental conditions, F. prausnitzii treatment was unable to minimize radiation-induced apoptosis of crypt epithelial cells in the colonic mucosa (Supplementary data S2). Nevertheless, we showed for the first time that its therapeutic benefit at 3 d is related to its ability to preserve mucosal self-renewal through the maintenance of an activated pool of stem/progenitor cells and of epithelial cell proliferation.

This work supports and offers some assumptions concerning F. prausnitzii mechanisms of action probably through an increase in some inflammatory parameters.

In several experiments using models of inflammatory bowel disease, one mechanism proposed to explain the therapeutic benefits of F. prausnitzii is its anti-inflammatory properties. 6,7,12 In our experimental context, in which colorectal irradiationinduced at 3 d a nonspecific immune response without a change in local pro-inflammatory cytokine production, we noted no anti-inflammatory effects after F. prausnitzii administration. Indeed, mucosal anti-inflammatory cytokine IL-10 was similar in control and irradiated animals pretreated or not by F. prausnitzii.

Nevertheless, we showed that in rats receiving F. prausnitzii, irradiation led to significant enhancement of resident macrophages. Macrophages play a key role in coordinating signals from luminal microbiota and injured colonic epithelium and thereby transmit regenerative inputs to colonic progenitor cells. 34,35 Recently, macrophages have been reported to be essential for the regenerative response of the intestine to lethal abdominal irradiation by rescuing LGR5+ intestinal stem cells. 35 In the context of radiation-induced dysregulation of colonic epithelial self-renewal, an increase of resident macrophages by F. prausnitzii treatment may play a role in the protection of the epithelial progenitor/ stem cell compartment and thus may help maintain colonic barrier integrity.

Moreover, in rats receiving F. prausnitzii, irradiation also induced up-regulation of the proinflammatory cytokines IL-1β, IL-18 by colonic mucosal cells. In a context in which the luminal level of F. prausnitzii could be increased, cell stress induced by irradiation should provide increased danger signals, which may stimulate NOD-like receptor (NLR) inflammasome signaling, leading to IL-1β and IL-18 activation and production by mucosal cells. It was previously reported that NLR inflammasome activation may be either regenerative or deleterious, depending on the cell type in which it is activated. 36 In response to colitis, acute or chronic NLR inflammasome activation in colonic epithelial cells and the subsequent production and secretion of IL-18 seem to be critical for maintaining microbiota stability, colonic homeostasis/regeneration, and barrier integrity against bacterial translocation, 37,38 whereas chronic NLR activation in immune cells like macrophages seems to have a pro-inflammatory effect. 36 The severity of colonic injury may be modulated by shifting the balance between protective and damaging effects depending on the inflammatory context and the experimental conditions. In our experimental conditions, we found that F. prausnitzii treatment after irradiation increases IL-18 production mostly by epithelial cells. So, in this context, the balance between regenerative and deleterious effects is probably in favor of protection via epithelial cell activation. Further experiments are needed to assess the relative impact of IL-18 produced by crypt epithelial cells in the protective effect of F. prausnitzii.

Finally, F. prausnitzii effects are also associated with its ability to preserve differentiated epithelial cells like Dclk1-positive tuft cells from radiation-induced loss. The use of Villin-Cre; Dclk1 flox/flox mice, which are specifically deficient in Dclk1 in the epithelial cells of the intestine, showed the involvement of this enzyme in the regeneration of the epithelium of the small intestine after whole-body irradiation. Moreover, Dclk1 mainly produced by the tuft cells in the epithelium seems to have a radioprotective effect on stem/ progenitor cells. In addition, in the absence of Dclk1 expression by tuft cells, irradiation-surviving stem/ progenitor cells appear to lose their ability to provide epithelial repair and Dclk1 deletion seems to have an impact on animal survival. 39,40 In the present study, the preservation of Dclk1-positive tuft cells by F. prausnitzii administration may have protected the colonic mucosa from radiation-induced damage. As chemosensory and secretory cells, tuft cells orchestrate the sensing of luminal content, the stromal immune response and regeneration of intestinal epithelial cells through the secretion of IL-25. 19,20,41 We showed that some tuft cells quantified in control rats or after irradiation in rats treated with F. prausnitzii also expressed IL-25 cytokine, whereas after irradiation alone they did not or did so rarely. This result points to the potential involvement of tuft cells in the prophylactic effects of F. prausnitzii on colonic mucosa self-renewal, through Dclk1 and/or IL-25 production. Further experiments are needed to demonstrate robustly the role played by tuft cells in the efficacy of F. prausnitzii against radiation toxicity.

Finally, in order to further our understanding with respect to the mechanisms of action of F.prausnitzii, we analyzed the microbiota composition through 16S rRNA sequencing. No effect of irradiation or F.prausnitzii administration was detected on microbiota richness (α-diversity). A deeper look into our results showed that only few rats, 4 from 48, were positive for F.prausnitzii and moreover, they were distributed among all the groups tested (control group, irradiated group and irradiated and F.prausnitzii treated group, data not shown). We also performed qPCR specific from F.prausnitzii but no amplifications were detected for any group using this method (data not shown). F. prausnitzii is Extremely Oxygen Sensitive and also very sensitive to pH 42 meaning that oral administration might lead to a massive death of the bacteria. We have already shown that it is indeed very difficult to colonize even germ-free mice with F.prausnitzii. 43 Rodents do not exhibit the same level of endogenous F.prausnitzii compared to humans which is one of the main difference between the two microbiota. 44 We have thus no evidence of F.prausnitzii colonization or even persistence in rats (this study) or in mice (chatel, personal communication) but there is still a protective effect for some parameters indicating that implementation is not required or it is at an undetectable level.

In a context of radiation-induced colonic damage, we have demonstrated for the first time that prophylactic F. prausnitzii treatment favors the reinforcement of the colonic mucosal barrier leading to radioprotective effects. We have established that F. prausnitzii prevents radiation-induced para-permeability and the associated acute nonspecific immune response, but also preserves epithelial integrity. Our results suggest that epithelial cell activation is involved in the benefits of acute F. prausnitzii treatment and that tuft cells could play a key role. Our study opens new perspectives in the protection of colonic mucosa from radiation toxicity using prophylactic F. prausnitzii treatment. New-generation probiotics might be a real hope in reducing acute side effects in patients who undergo pelvic radiotherapy. It has also been suggested that the severity of acute radiation toxicity may predict the severity of chronic symptoms, known as consequential late damage. 45 Therefore, F. prausnitzii-induced acute protection may be beneficial in reducing late side effects. Clinical use of F. prausnitzii could help improve the quality of life of these patients.

Resume

Effet d'un transfert du microbiote fécale sur les atteintes épithéliales radio-induite du côlon de rat. La radiothérapie représente 60 % des stratégies thérapeutiques du cancer de la région pelvienne. Les conséquences de l'irradiation dans les tissus entourant la tumeur peuvent apparaître plusieurs années après la radiothérapie. Ceci a été décrit en 2010 comme une nouvelle pathologie : la "Pelvic Radiation Disease". Au laboratoire, l'objectif est de développer de nouvelles stratégies thérapeutiques pour réduire la toxicité tissulaire induite par la radiothérapie. Des données cliniques prospectives ont démontré que pendant et après la radiothérapie pelvienne, il y a un changement dans le microbiote en parallèle des complications intestinales. Ceci pourrait être décrit comme une dysbiose, une perturbation de la relation de symbiose entre l'hôte et le microbiote intestinal. Notre objectif est d'attester, dans un modèle préclinique pertinent, que l'irradiation conduit à des modifications de l'écosystème intestinal qui in fine est responsable de l'évolution des lésions. Nous avons développé un modèle d'irradiation localisée approprié utilisant des doses fractionnées (très similaires à la procédure clinique) pour obtenir des protocoles d'irradiation qui induisent des dommages épithéliaux modérés ou sévères. En collaboration avec l'Institut national de recherche agronomique et environnementale, nous avons analysé la dynamique temporelle (i) de l'altération du microbiote fécal (séquençage métagénomique Shot Gun) et du métabolome (méthodes analytiques basées sur la chromatographie liquid couple à de la spectrométrie de masse) et (ii) de la réponse immunitaire et épithéliale de l'hôte par immunomarquage. Nous avons rapporté qu'en fonction du protocole utilisé, l'irradiation augmente la diversité du microbiote, stimule la réponse immunitaire de l'hôte et altère les tissus au fil du temps. Nous avons également démontré que le transfert de microbieote fécale réduit la toxicité colique uniquement chez les animaux présentant des dommages épithéliaux modérés. Les traitements existants pour la PRD ne sont que symptomatiques, c'est pourquoi cette étude est d'un intérêt clinique primordial.

Faecal microbial transplantation to reduce radiation-induced colonic epithelial damages: application to Pelvic Radiation disease. Radiotherapy accounts for 60% of treatment strategies for pelvic cancer. The consequences of radiation in the tissues surrounding the tumour can appear several years after radiotherapy. This was described in 2010 as a new pathology: Pelvic Radiation Disease (PRD). The laboratory aims to develop new therapeutic strategies to reduce the tissue toxicity induced by radiotherapy. Prospective clinical data have shown that during and after pelvic radiotherapy, there is a change in the microbiota in parallel with intestinal complications. This could be described as dysbiosis, disrupting the symbiotic relationship between the host and the gut microbiota. In a relevant preclinical model, we aim to demonstrate that irradiation leads to changes in the gut ecosystem which ultimately is responsible for the evolution of lesions. We have developed a suitable localised irradiation model using fractionated doses (very similar to the clinical procedure) to obtain irradiation protocols that induce moderate to severe epithelial damage. In collaboration with the Institut National de Recherche Agronomique et Environnement, we analysed the temporal dynamics of (i) the alteration of the faecal microbiota (Shot Gun metagenomic sequencing) and the metabolome (analytical methods based on liquid chromatography-mass spectrometry) and (ii) the host immune and epithelial response by immunostaining. We reported that, depending on the protocol used, irradiation increases the diversity of the microbiota, stimulates the host immune response and alters the tissue over time. We have also shown that faecal microbiota transfer reduces colonic toxicity only in animals with moderate epithelial damage. Existing treatments for PRD are only symptomatic, so this study is of primary clinical interest.

Figure 1 .

 1 Figure 1. Anatomy of the pelvic area in women (top) and men (bottom) (2).

Figure 2

 2 Figure 2. Incidence rate (y-axis) for each highest incidence pelvic area cancers (x-axis) (3).

Figure 3 .

 3 Figure 3. Stages of the life cycle of a cell including G1, S, G2 phs (5).

Figure 4 .

 4 Figure 4. Details of the various stages of mitosis (6).

  5), while proton radiotherapy needs a particle accelerator (cyclotron or synchrotron) (Figure 6.) B. Application of radiotherapy in the context of pelvic cancers a. Radiotherapy objectives b. Type of radiotherapy and energy available for radiotherapy i. EBRT (15,16)

Figure 6 .

 6 Figure 6. Example of cyclotron for medical purpose (16).

Figure 5 .Figure 5 .

 55 Figure 5.Figure5. Example of a medical linear accelerator used for EBRT(15).

Figure 6 .

 6 Figure 6.

Figure 7 .

 7 Figure 7. Examples of dose distribution for different types (3D-CRT, IMRT, VMAT, PSTP, IMPT and BT) of radiotherapy delivered in the context of prostate tumours(38).

  particles g-rays 73.8 days 0.670 (b particles),0.38 (g-rays) Cobalt 60 b particles g-rays 5.26 years 0.318 (b particles), 1.25 (g-rays) particles g -rays 30 years 0.51 (b particles), 0.662 (g-rays) (40,41) A new type of radiotherapy is gaining popularity among clinicians, especially for treating pelvic cancer: radiopharmaceuticals. Radiopharmaceuticals are drugs containing a radioactive substance delivered to the tumour directly or by a vehicle reaching the target due to its physicochemical properties. Compared to EBRT techniques, radiopharmaceuticals do not deliver the same amount of radiation to all cells

Figure 8 .

 8 Figure 8. Configuration of the treatment plan with a CT scan in the axial plane. (A) and coronal plane (B) for cervical cancer patient. Dose-volume histogram describing organ dose exposition for clinical target (red), bladder (purple), rectum (green) and sigmoid (yellow) (41).

Figure 9 .

 9 Figure 9. Difference of tumour cell distribution of radiation beams between (a) EBRT and (b) radiopharmaceutical (43).

  sensitivity to irradiation. The linear-quadratic model is a mathematical modelling of an in vitro cell survival curve (S) as a function of dose (d) S=e-(ad+bd 2 ). This model integrates two components, a component a proportionally to the dose and a component b proportionally to the square of the dose and allows to extract the ratio a/b which is of some interest in clinical practice. The ratio a/b is a dose expressed in Gy and corresponds to the dose for which the cell death related to the linear component a (directly lethal events) is equal to the death related to the quadratic component b (accumulations of sub-lethal events). The advantage of this model

Figure 11 .

 11 Figure 11. Explanation of grade of CTCAE for diarrhea and colitis (left) and RTOG (right) concerning toxicity to the bowel related to anti-cancer drug and irradiation[START_REF] Chinnachamy | Evaluation of Interobserver and Interscale Agreement in Assessing Late Bowel Toxicity after Pelvic Radiation in Patients with Carcinoma of the Cervix[END_REF][START_REF] Parente | Clinic, Endoscopic and Histological Features in Patients Treated with ICI Developing GI Toxicity: Some News and Reappraisal from a Mono-Institutional Experience[END_REF].

  mucosa is composed of the epithelium in which lamina propria and muscularis mucosa differ from other organs of the digestive tract. It is the part most exposed to the intestinal lumen. The epithelium of the colon is composed of Lieberkühn crypts and has two main roles: to absorb substances of interest and to retain from the intestinal lumen what might be toxic or unnecessary for the tissues. The crypts are the main component of the mucosal barrier. Each crypt is organised in the same way. At the base of the crypt are mainly undifferentiated cells, the stem cells, which is why it is called the stem cell niche. Stem cells can be quiescent or active (differentiated according to the speed of their cell cycle). Active stem cells, because of their rapid cell cycle, are more sensitive to any toxic a. Mucosa i. Epithelium:

Figure 12 .

 12 Figure 12. Structural organisation of the large intestine. Details of the different layers of the colonic tissue (66).

Figure 13 .

 13 Figure 13. Differentiated colonic epithelial cells. A. enterocytes with all protein junctions contained in the epithelial cells described (zonula occludens, adherens, macula adherens, communicans) (68). B. goblet cell with all inner organelles detailed (69). C. example of an enteroendocrine cell with the process of GLP1 production and its destination[START_REF] Cummings | Gastrointestinal regulation of food intake [Internet[END_REF]. D. Tuft cell with all its constituents and how it is positioned in relation to the neighbouring cell[START_REF] Hendel | Tuft Cells and Their Role in Intestinal Diseases[END_REF].

  macrophages are Classically Activated Macrophages (CAMµs) known as M1 macrophages, Alternatively Activated Macrophages (AA-Mµ) or M2 macrophages and Regulatory Macrophages (RMµ). CAMµs are involved in pro-inflammatory and defensive action, AA-Mµ in repair and wound healing, and RMµ in anti-inflammatory action. The phenotype of macrophages depends on the signal received by the resident macrophages or monocytes. Thus, to be differentiated into CAMµs, IFN, oligosaccharides (LPS) or ligands to the Toll-Like Receptor (TLR) are required, TLRs recognise the element of microorganisms and are an important element in innate inflammation. AA-Mµ could be divided into four subsections: M2a, M2b, M2c and M2d. M2a becomes so due to the presence of interleukins 4 and 13 (IL-4, IL-13), M2b due to TLR immune complex or interleukin 1 (IL-1) agonist, M2c due to interleukin 10 (IL-10), TGFb or glucocorticoid and M2d which are tumour-associated macrophages are initiated with the combined exposure of TLR activation and adenosine A2 receptor agonist and or interleukin 6 (IL-6). Finally, exposure to TLR ligands, such as prostaglandin and adenosine, allows differentiation into RMµ (Figure 14.) (72,73). During inflammation, M1s only switch to the M2 phenotype if neutrophils are present; otherwise, polarisation does not occur.

Figure 14 .

 14 Figure 14. Description of the function of macrophages according to type and subtype and which mediator promotes which phenotype.

Figure 15 .

 15 Figure 15. All subtype of ILC reuniting NKc ILC1, ILC2, ILC3 and LTi and their roles.

Figure 16 .Figure 17 .

 1617 Figure 16. Description of all subtypes of LT and LB thus their different actions

Figure 19 .

 19 Figure 19. Example of different symbiosis type. A. Barnacles living on whales are an example of a commensal relationship that benefits only the barnacles, with the whales providing habitat and nutrients without receiving any benefit in return (86). B. Mutualistic relationship between buffalo and red-billed woodpecker. The bird feeds on the buffalo's parasite and warns the buffalo of the predator's approach by flying or calling (87). C. A mutualistic relationship called a lichen, which involves algae or cyanobacteria providing elements from photosynthesis to a fungus, while these fungi protect them from the environment (88). D. In the mutualistic relationship between the clownfish and the anemone, both are sources of nutrients for the other and protect each other from their respective predators (89). E. The cactus mistletoe is a parasite of the cactus, it benefits from the nutrients given by the cactus, while infection of the cactus by Tristerix aphyllus results in a decrease in its reproductive rate and photosynthesis (90). F. The parasiteOphiocordyceps unilateralis controls the infected ants and forces them to move to a place where the humidity is favourable for the growth of the fungus, and then the ants wait to die while the fungus grows and produces spores that can infect other ants[START_REF]How a parasitic fungus turns ants into 'zombies[END_REF].

Figure 20 .

 20 Figure 20. Stages of Sanger sequencing methods A. Sequencing phase where the DNA polymerase, starting from the primer that hybridises to the template DNA fragment, elongates each DNA sample with deoxynucleotides that allow elongation and dideoxynucleotides that stop it. B. Electrophoresis step where each DNA fragment migrates through a polyacrylamide gel (PAGE) and each fluorochrome is excited by a laser. C. The detector transmits the signal received by each fluorochrome of the different fragments to a computer where, thanks to specific software, the sequence is analysed and the template DNA fragment is reconstituted (92).

  Primers are attached to each fragment, and the amplification and elongation step begins. Each sequencer uses a technique of its ones. Roche 454 cut DNA or RNA into "read" with an average size of 400 base-pair (bp) and used pyrosequencing (emission of light) to detect nucleotide in a DNA or RNA fragment (Figure 21.A.). Concerning Illumina, "read" size comes from 50 to 300bp, and they use the fluorescence technic as the Sanger method to detect specific dNTP and corresponding nucleotide (Figure 21.B.). Finally, the Ion torrent cuts DNA or RNA into fragments from 200 to 400bp and detects nucleotides by measuring pH (Figure 21.C).

Figure 21 .

 21 Figure 21. Second-generation of sequencer.A. Different steps in the Roche 454 sequencing technique. First, the DNA or RNA strand is fragmented, then the primers are bound to both sides of the reads and each primer is attached to its specific beads. The next step is emulsion pcr where each read on the bead is amplified and attached to the bead in the same way. The beads are then placed on a picotitration plate so that sequencing can begin, a light is emitted when a specific dNTP is bound to the complementary strand, allowing identification of the nucleotides and the order in which they occur in each read[START_REF] Siqueira | Pyrosequencing as a tool for better understanding of human microbiomes[END_REF]. B. Description of the Illumina sequencing process with the fragmentation of the DNA or RNA strand into 150-300 bp reads, hybridisation with primers on either side of the reads and then onto complementary primers on the flow cell. Then the Production of a complementary read that binds directly to the primer on the flow cell is performed followed by a first clean-up step that removes the first reads that have bound to the flow cell. A bridge is formed by the second primers attached to the reads that have bound to their complementary primer on the flow cell, followed by bridge amplification forming clusters for each read.A further clean-up step is performed to cleave one of the read strands, the next step is the start of sequencing, the polymerase allows specific dNTPs coupled to a fluorochrome to be attached to the primer and a laser is turned on to detect the fluorochrome whose emitted wavelength allows identification of the nucleotide to which it is attached, thus the sequence of the reads is determined[START_REF] Chaitankar | Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research[END_REF]. C. How the Ion Torrent sequencer works. The first step is to create a DNA or RNA fragment and to ligate the primers on both sides of each fragment. One of the two primers for each read will be attached to a bead and a PCR will be performed to amplify the number of identical reads on each bead. Then each bead will be placed in a well of the micromachine with one bead per well. A solution containing a polymerase and a specific dNTP is incorporated into each well, causing the initiation of complementary strand production. When the specific dNTP in the solution is bound to the DNA or RNA template, the reaction produces the formation of H+, resulting in a pH change in the solution. Each well is connected to a pH meter and so when the pH changes, the pH meter detects this and records the information of the pH change when a specific dNTP is added to the solution. A clean-up process is carried out between each incorporation of a new specific dNTP to remove the excess of previous dNTPs. When a single dNTP is added, it produces a limited amount of H+ and the more dNTPs that are added, the more H+ are produced and thus detected. Thus, if the reading contains a repeat of the same nucleotide several times in a row, it will be detected by the pH meter[START_REF] Golan | Using state machines to model the Ion Torrent sequencing process and to improve read error rates[END_REF].

Figure 22 .

 22 Figure 22. Operation of third generation sequencers.A. How PacBio technology works, with a first step of fragmenting the DNA or RNA and then ligating these fragments to primers on both sides. Then, each fragment is incorporated into a SMRT consisting of millions of ZMWs, each of which will host a fragment. The sequencing process can then begin by incorporating a polymerase and a specific dNTP coupled to a fluorochrome into each ZMW. The attachment of the fluorochrome to the complementary label results in the emission of light which is detected and recorded[START_REF] Metzker | Sequencing technologies -the next generation[END_REF]. B. Operation of the Oxford Nanopore sequencer. A nanopore protein coupled to a helicase is embedded in an electrically resistant membrane which is connected to a detector chip just below it. The DNA or RNA is fragmented into long reads and ligated with adapters on both sides, and these reads find their specific adapters in the nanopore. Then the single strands of DNA or RNA pass through the nanopore, each nucleotide passing through inducing a compositionspecific change in the ionic current that is detected by the sensor chip (100).

  ii. Prediction of microorganisms' function iii. The cost C. The deepening of knowledge with sequencing ecosystem or an entire environment has already been developed. Still, the tools to calculate it needed to be improved. The a-diversity is the diversity of different taxa present in a sample. It is characterised by the number of said taxa (richness) and/or the number of taxa with details of their abundance (distribution). Different indices can be used to calculate a-diversity, such as Chao's index (based on the number of different taxa in a sample and with a prediction on which are rarer) and Shannon's index (which takes into account the number of taxa and their abundance in the sample). The b-diversity represents the similarity between two samples' subpopulation based on microbial composition. Like a-diversity there are several ways to represent b-diversity. If we consider the genetic distance between each species present in two different samples, the best way is to use the Unifrac distance. The Bray-Curtis method is the most suitable for comparing the same taxa's abundance in two samples. Finally, Jaccard's index should be used to calculate the distance solely based on the presence or absence of a taxon in each sample. All dissimilarity distance indices are between 0 and 1, Depending on the index used, 0 may represent a completely common or completely different microbiota; a Unifrac weighted distance of 0 means that the two samples have the same composition concerning the branch of the phylogenetic tree, a Bray-Curtis distance of 0 means that the microbial composition of the two samples shares the same taxa in the same abundance, and on the contrary a Jaccard of 0 shows that the samples have the different taxa present. Finally, gdiversity reflects the diversity in an entire environment composed of different ecosystems population. It, therefore, considers the whole a-diversity of each ecosystem. Before

Figure 23 .

 23 Figure 23. Example of a phylogenic tree.This phylogenic tree comes from dataset comprising 13,133 human gut microbiome analyses from 75 different studies. This tree shows many species, most of which are unidentified metagenomic species (UMGS) compared to those found in the human gut reference (HGR). The outer layer (green bar) represents the genome prevalence in the 13,133 datasets. The gut microbiota shows great diversity within and between individuals. It is therefore important to be representative of as many people as possible, and currently people from Africa and South America are under-represented in microbiota studies. This tree also highlights the fact that it is difficult to interpret metagenomic studies when many species are still unknown. It should also be considered that the majority of microbiota studies focus on bacteria, and moreover on the most abundant species, which can create biases in the interpretation(102)

  they arrive in the ileum, 95% of the primary acids are reabsorbed and return to the liver, where they are recycled. They can also undergo deconjugation, their glycine or taurine is eliminated, thanks to the intestinal microbiota, and then become secondary bile acids which can either be reabsorbed or eliminated in the faeces. One of the secondary bile acids, deoxycholic acid, has been shown to play a role in inhibiting the growth of the pathogenic bacteria Clostridium difficile (111). Researchers have shown that indole derivatives can also play a role in tissue protection by strengthening the barrier, such as ligands of the aryl hydrocarbon receptor (AhR), such as 6-formylindolo(3,2-b) carbazole (FICZ), which is a product of tryptophan metabolism (i). Competition (ii). Metabolites production effect on epithelial cells by intestinal microorganisms. Activation of the AhR by FICZ ligation, administered as a treatment to mice in a DSS model, increases the expression of tight junction (TJ) proteins. The action of FICZ was confirmed in vitro. Exposure of Caco-2 cells to tumour necrosis factor

  polyamines or bacterial vitamins. One of the BCAAs, isoleucine, contributes to maintaining a proliferative state of Treg. This study first demonstrated that mice fed a diet low in BCAAs had a reduced number of FOXP3+ Tregs in the spleen. Ikeda et al. examined the effect of isoleucine on Treg cells. Authors then exposed naive LTCD4 cells to TGF-b (which helps transform naive LTCD4 cells into Tregs) with or without isoleucine and found that isoleucineFrom host From microorganisms

  [START_REF] Sonis | Perspectives on cancer therapy-induced mucosal injury[END_REF] described the course of oral mucositis triggered by exposure of healthy tissue to radiation. Thus, they establish from literature data that mucositis of the intestinal tract occurs in five phases. The initiation of the process is triggered by the encounter of reactive ions with water molecules inside the cells producing ROS (131). ROS cause DNA breaks, leading to mucosal and endothelial cell death. ROS can also activate the transcription factor NF-kB, which positively regulates genes, notably those involved in the synthesis of pro-inflammatory cytokines, in the activation of the cyclooxygenase 2 (COX2) pathway, and in the expression of adhesion molecules, which leads to angiogenesis. Next comes an amplification of inflammation, and apoptosis occurs, which leads to tissue damage, including ulceration formation. Finally, the healing phase can lead to excessive proliferation, causing fibrosis driven by TGF-b1 and causing long-term consequences (132). This process has been described in oral mucosa, but as authors precise in the paper, there is no reason to think it happened differently in the gut mucosae (133).

  depending on the patient, their cancer and treatment, and their gender. The study ofFerreira et al., involving only men, showed an increase in this Chao index, but no information where given concerning the Shannon index, compared to the two studies ofWang A et al., and Wang Z et al., where the patients included were predominantly or exclusively women and where an opposite trend was shown with a decrease in Chao index (for the second one only) and Shannon index (for both) overtime after radiotherapy and more marked in patients with more severe symptoms. Gender and associated microbiota could be a factor in the different responses to radiotherapy treatment, as it is suggested in this experimental study (139).Other clinical studies have investigated the effect of pelvic radiotherapy on the gut microbiota.Still, they did not exclude patients who received chemotherapy during radiotherapy treatment or antibiotics before or during radiotherapy treatment. It introduces a bias in the study regarding the link between exposure of non-cancerous tissue to ionising radiation and alteration of the gut microbiota and the development of PRD, as chemotherapy and antibiotic agents have been shown to affect the gut microbiota with generally a decrease in a-diversity. These studies showed a reduction in a-diversity after the patient had received all these treatments. (140-142).As each study involves a small number of patients, it is difficult to demonstrate a specific change in the gut microbiota composition after pelvic radiotherapy. As clinical data is difficult to obtain, it is interesting to see what happens in animals. Experimental studies can help to get a clearer picture of the role that gut microbiota might play in the development of radiationinduced gut toxicity.

  conventional treatments would be interesting to see what probiotics could bring compared towhat is already on the market.Faecal microbiota transfer is another solution that can provide beneficial microorganisms to the host. This treatment has the advantage that it does not depend on knowledge of culturomics and also has a greater chance of the microorganisms taking hold and having an effect. In contrast to probiotic studies, Faecal Microbiota Transfer (FMT) studies are conducted in a curative setting in patients with chronic PRD.The transfer of faecal microbiota in the context of localised pelvic irradiation has not been the subject of any experimental studies. Still, one clinical study has been carried out, and two case reports have been published.The clinical study includes 5 patients who received pelvic radiotherapy for gynaecological cancer and subsequently developed PRD. Depending on the case, the patients received from one to three faecal microbiota transfers. The FMTs were derived from young, healthy donors and were delivered via nasojejunal tube. For these patients, the median time from the end of their radiotherapy treatment to enrolment in the clinical trial was 21 months; no information was provided regarding the time of their diagnosis of PRD. The reported efficacy of FMT treatments appears to depend on two factors: the age of the patient and the severity grade of their disease. In three out of five patients, FMT reduced the severity of their PRD by at least one grade. The other two patients who did not improve their disease were the oldest in the study, and one had the most severe disease of the five patients recruited. Several hypotheses can be deduced from these results, the first concerning the match between donor and recipient.

  the glands, and a diagnosis of radiation proctitis was made. The patient was then given 4 faecal microbiota transfers from 8 different donors aged between 21 and 24 years and three samples were mixed for each transfer prepared. The FMT was administered in two different ways. The first two transfers were deposited in the lower intestine while the next two were deposited in the middle of the intestine as it was difficult to go further by endoscopy. A maximum of 1 hour elapsed between each donor collection and the transfer to the patient. The FMT was administered every day for three days almost once a month (March, April, May and July). Six months after treatment the patient's symptoms (hematochezia, diarrhoea, red and mucous stools) were improved and her RTOG score improved from 2 to 1. Colonoscopy showed that inflammation of the sigmoid and rectal mucosa was reduced. In addition, a microbiota measurement was performed. Patient's stool was recovered before treatment, 28 days after the first FMT, 30 days after the second and 70 days after the third. rRNA sequencing was performed on all samples as well as on the mix used for the first FMT. a and b diversity increased during FMT treatment. The majority phylum before FMT was Firmicutes and became Bacteroidetes after. Thus, the composition of the patient's faecal microbiota during FMT approximated that of the donors. This study demonstrates that multiple treatments with FMT from a donor pool may allow the microbial composition of the recipient to be shifted as well as improve the patient's quality of life. It is difficult to establish correlations as this is a single-patient study, but it indicates the interesting potential that FMT may have in the context of PRD that is not too severe in grade (159).

June 14 th 6 Stéphane Grison 7 ,Fabien Milliat 1 ,

 14671 Time-course faecal microbiota and metabolome modifications in a new preclinical "pelvic radiation disease" rodent model: a comprehensive, integrated study. Mallia Geiger 1,2 , Oscar Gitton-Quent 3, Mohamed Amine Benadjaoud 4 , Maâmar Souidi Jean Charles Martin 8 , Jean-Marc Chatel 5 , Joël Doré 3,5 , Mathieu Almeida 2 , Alexandra Sémont 1* . 1 Institut de Radioprotection et de Sûreté Nucléaire (IRSN)/PSE-SANTE/SERAMED/LRMed, Fontenay-aux-Roses, France 2 Université Paris-Sorbonne 3 Université Paris-Saclay, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'environnement (INRAE), MetaGenoPolis, Jouy-en-Josas, FranceIntroductionThe healthy adult intestine contains around 10 12 microbes/g. The microbiota comprises viruses, fungi, archaea and some 50 trillion bacteria living in symbiosis with the host. The symbiotic relationships between the gut ecosystem's microbiota and host play a fundamental role in intestinal homoeostasis and tissue regeneration in response to injury. Commensal bacteria involvement in intestinal health or disease is currently more documented. Under physiological conditions and symbiosis, one of the functions of the commensal bacteria is to prevent intestinal pathogen colonisation by direct physical or chemical competition. Another mechanism that allows this function is their ability to reinforce the epithelial barrier integrity by acting on the epithelial cells and the immune response. Environmental modifications induced by diet, antibiotics, circadian rhythm, xenobiotics, or therapies like chemo-, immuno-or radiotherapy, could lead to gut microbiota disorders, including commensal bacterial richness reduction and increase in pathobionts. Modified microbiota provoking altered mutualistic relationships with the host could be followed by a loss of intestinal barrier integrity, bacterial translocation and inflammatory response that could, in turn, modify redox balance and, in a vicious cycle, further aggravate microbiota alterations[1,2] Depending on stress conditions, i.e., intensity and/or duration, deregulated reciprocal relationships with auto-exacerbating signals from each other could prevent physiological ecosystem resilience and its return to a basal stable state. This leads to a critical transition from symbiosis to an altered stable state called dysbiosis, without the ability of reversion, even if stresses are stopped. Dysbiosis could be characteristic of predisease or chronic disease[3][4][5] as suggested for autoimmune or autoinflammatory disorders such as inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis), metabolic disease (e.g., type 2 diabete or obesity) neurological conditions (e.g., Alzheimer's, autism or Parkinson's), but also several cancers.

  IRSN animal facility is accredited by the French Ministry of Agriculture for performing experiments on rodents. 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 Ethics Committee #81 (approval number E92-032-01) and authorized by the French Ministry of Research under the reference APAFiS#23843-2020012816544712v1.

  For macrophages: a Mouse anti-rat CD68 (AbDserotec ref: MCA341R) diluted respectively at 1/35 and 1/200 dilution in Diluent Antibody (Diagomics Zytomed Systems #ZUC025-100). Slides were left for 1h in a 37° incubator and then rinsed with TBST (2X) and TBS (1X). The secondary antibodies used in both protocols were polymer HRP, an anti-rabbit (Diagomics GBI labs #D13-110) for neutrophils and an anti-mouse (GBI labs ref: D55-18) for macrophages. Once deposited, the slides were left for 30 minutes at room temperature and then rinsed in TBS. Then the Permanent HRP Green substrate kit (Diagomics Zytomed Systems #ZUC070-100) was applied to the slides. The slides were left for 10 minutes in the dark with the substrate. A counterstain with Nuclear Fast Red (Vector ref: H-3403) was performed for 10min then the slides are rinsed and placed in an automated system (PRISMA) which carried out the dehydration and mounting of the slides. The negative control was assessed with rabbit and mouse IgG (Dako, X0931) instead of the analysed primary antibodies.

  The statistical analysis for ulcer and immune cell kinetics is based first on Shapiro-Wilk normality tests and variance equality. Student's, Welch or Wilcoxon tests were performed depending on Shapiro-Wilk and variance results. The results are represented in the form of a boxplot and barplot. Statistical significance was considered when the p-value was < 0.05.Significance can be indicated by stars (the number of stars represents the power of the p-value) or letters. Boxplots or barplots with the same letters are similar. The letters are coloured to indicate differences within a single group, with each colour representing a group (e.g., CTR:

  Figure 1

  Fig.2 Temporal effect of 3X10 Gy and 3X14 Gy fractionated colorectal irradiations on innate inflammatory response in the colonic mucosa (days 14, 28 and 56 post-irradiation). A Representative pictures of colonic sections immunostaining by MPO (for neutrophils), CD68 (for macrophages) or of staining by toluidine blue (for mast cells) in control (CTR) animals, 3X10 Gy or 3X14Gy irradiated animals at days 14 and 56. B-E Boxplots show B colitis score including 1) mucosa inflammatory cell infiltration 2) submucosal inflammatory cell infiltration, submucosa oedema, colitis cystica profunda, and extracellular matrix deposition, C neutrophils, D macrophage and E mast cell infiltration at D14, D28 and D56 post-irradiation. Boxplots represent the median (Q3/Q1) score or number of immune cells per unit of area (µm2 ). n= 6 rats/group at D14 and D28 and n=12 rats/group at D56. Significances indicated between group (*) and time (letter) were calculated with Student, Welch or Mann-Whitney test according to normality and variance equality* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Boxplots sharing the same letter and colour letters are not different.

Figure 2

 2 Figure 2

Fig. 3

 3 Fig.3 Time course of rat faecal microbiome following colorectal fractionated irradiation at 3X10 Gy and 3X14 Gy doses. Quantitative assessment of shotgun metagenomics of 28 samples was analysed with n=12 rats per group with 8 times points, i.e. days 0,3, 7, 14, 21, 28, 42,[START_REF] Andreyev | Defining pelvicradiation disease for the survivorship era[END_REF]. A B Alpha-diversity analysis based on A the Shannon index and B the richness (number of species within samples). C PCoA of faecal bacterial communities based on the dissimilarity distance. D E Boxplot of dissimilarity distance as a function of irradiation dose D and time E. Concerning alpha diversity, Student, Welch or Wilcoxon tests were realised according to the respect of normality and variance equality. Differences in theShannon index between the 3x14Gy and CTR groups were seen on days 7 and 14. No difference over time for each group was detected. For the richness, the 3x10Gy differed from the CTR and the 3x14Gy on day 3 and the 3x14Gy only on day 28. Over time the 3x10Gy group showed differences between days 3,21, and 28 compared to day 56. Significances indicated between group and time on boxplots were calculated with permutational multivariate analysis of variance with permutation = 100000, and correction of p-value was realised with Benjamini-Hochberg correction. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Figure

  Figure 3

Fig. 4

 4 Fig.4 Dynamic modulation clustering of A the 2 Phyla identified with a temporal modulation in the 3X10 Gy group vs control (CTR) group. B the 66 species identified with a temporal modulation in the 3X10 Gy group vs CTR group. C the 5 phyla identified with a temporal modulation in the 3X14Gy group vs CTR group. D the 155 species identified with a temporal modulation in the 3X14Gy group vs CTR group. n=12 rats per group with 8 times points, i.e. days 0,3,7,14,21,28,42,56.

Fig. 5

 5 Fig. 5 Time course of rat faecal metabolome following colorectal fractionated irradiation at 3X10 Gy and 3X14 Gy doses. Quantitative assessment of metabolites of 288 samples was analysed with n=12 rats per group with 8 times points, i.e. days 0,3, 7, 14, 21, 28, 42, 56. A PCoA of faecal metabolites based on the Bray-Curtis dissimilarity distance. B C Box-plot of Bray-Curtis dissimilarity distance as a function of irradiation dose B and time C. Significances indicated between group and time on boxplots were calculated with permutational multivariate analysis of variance with permutation = 100000, and correction of p-value was realised with Benjamini-Hochberg correction. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 D Examples of dynamic profiles for 4 metabolites both for the control (CTR) group and 3x14 Gy group. CI : Confident Interval. The green line represents the time interval with a significative difference between the groups for the metabolite analysed. This was done for 208 metabolites identified in the data set. E Clustering results from the dynamic modulation of 89 metabolites identified from the comparison between CTR and 3X14 Gy groups.

Figure 5

 5 Figure 5

Figure 6 AFig 6

 66 Figure 6A

day 25 to 50 .

 50 Significant increases over almost the entire time frame between days 3 and 56 compared to the control group were only observed for proline (Supplementary Fig S6). The list of functional bacterial modules by clusters, from the multiscale clustering study, showed that faecal glutamate variation after irradiation is associated with in silico modulation of many bacterial functional modules as glutamate degradation (4-aminobutanoate)/ aminobutyric acid (GABA) synthesis, glutamate synthesis II, glutamine degradation (oxoglutarate or ammonia pathway) and histamine degradation (formiminoglutamate pathway) (Clusters 3_X_scale and 6_x_scale, Fig 7A and 8).. The temporal evolution of faecal arginine and ornithine matches with in silico increase of biosynthesis functional module (through ornithine or glutamate pathways respectively) (Cluster 4_x_scale and 8_x_scale, Fig 7A and 8). In silico proline biosynthesis and degradation (glutamate pathway) mirrors temporal dynamics (clusters 4_x_scale and 6_x_scale, respectively, Fig 7A and 8).

Fig 8

 8 Fig 8Completeness analysis 1 done in silico within each cluster (2 to 8) was generated by the multiscale clustering approach. Results are shown for clusters 2 to 8 because no completeness has been identified in cluster 1. This approach allowed us to identify bacterial metabolic functional modules, i.e., matching with certain communities of bacteria in the same cluster and identifying putative functions linked to these bacteria.

  Figure 8 Cluster 1 x_scale Cluster 4_ x_scale Cluster 2_x_scale Cluster 3_ x_scale No completeness found.

of 4

 4 temporal patterns show positive correlations, common to moderate (Clusters A_bfm to D_bfm) and severe lesions (clusters E_bfm to H_bfm). Indeed, clusters B_bfm and F_bfm show an early and transient positive correlation, clusters A_bfm and E_bfm a transient positive correlation at a temporal intermediate period (between days 20 and 50) and clusters D_bfm and H_bfm a gradually increasing positive correlation over time. Cluster C_bfm shows a positive correlation between moderate lesions and the temporal evolution of functional bacterial modules reported for the latest time-points (from D40).

Fig 10

 10 Fig 10 Metabolic pathway networks created with pathway function from MetaboloAnalyst software. In red are highlighted metabolites modulated in the 3x14 Gy group vs control (CTR) group, i.e. glutamate, arginine, ornithine and proline previously identified from the multiscale approach but also creatinine, sarcosine, putrescine and spermidine.

Figure 10

 10 Figure 10

Figure 25 .CFigure 26 .Figure 27 .Figure 28 .

 25262728 Figure 25. Experimental design for protocol 01. Autologous FMT was performed intragastrically. Treatment began the day of the last irradiation dose and was repeated four times on day 4 (D4), day seven (D7) and day 11 (D11); sacrifice time was on day 14 (D14)

(Figure 29 .Figure 30 .

 2930 Figure 29. Experimental design for protocol 05. A.B. Autologous FMT was performed intragastrically. A.Treatment began on D3 lasted 4 days until day 6 (D6). Sacrifice time was on D7. B.Treatment began on D10 lasted four days until day 13 (D13). Sacrifice time was on D14.

Figure 31 .

 31 Figure 31. Experimental design for protocol 07. Autologous FMT or pooled faeces were administrated intragastrically. Treatment began on the day of the last irradiation dose and was repeated on D4, D7 and D11. Sacrifice time was on D14.

Figure 32 .

 32 Figure 32. Size of the lesion according to the degree of severity in protocol 01. A. Lesion for the groups CTR, 3x10Gy NaCl-and 3x10Gy FMTa.B. Lesion for the groups CTR, 3x14Gy NaCl-and 3x14Gy FMTa.C.E. Each measurement was made on groups CTR, CTR NaCl-, 3x10Gy NaCl-and 3x10Gy FMTa. C. Number of neutrophils per unit of area. Number of animals per MPO intensity scores that goes from 0 (low intensity) to 2 (hight intensity). E. Number of macrophages per unit of. F. Number of mastocytes per unit of area for. P-valuewere indicated as followed: * : p.value <0.05, ** p.value <0.01, *** : p.value <0.001, **** p.value <0.0001.

  3x10Gy FMTa group respectively (p-value <0.0001). The size reduction in the FMTa group compared to NaCl-was such that even a reduction in total size was seen (median of 21408.5 µm for 3x10Gy NaCl-and 18705.03 µm for 3x10Gy FMTa group) (p-value <0.05) (Figure.32.A) which was not the case for 3x14Gy irradiation protocol groups (Figure.32.B) for which FMTa did not ameliorate any lesion type.

  groups. Even more, in the worst case, it deteriorates the tissues more than NaCl-administration; for protocol A, the ulcer size from the animals treated with NaCl was smaller than in animals treated with FMTa. (median size of ulcer of 13156, 19 µm for the 3x14Gy NaCl-group and 16449,815 µm for the 3x14Gy FMTa group) (p-value <0.05) (Figure.33.A).

Figure 33 .

 33 Figure 33. Size of the lesion according to the degree of severity in protocol 02. A. Lesion for the groups CTR, 3x14Gy NaCl-and 3x14Gy FMTa time of sacrifice was D3. B. Lesion for the groups CTR, 3x14Gy NaCl-and 3x14Gy FMTa time of sacrifice was D7. C. Lesion for the groups CTR, 3x14Gy NaCl-and 3x14Gy FMTa time of sacrifice was D10. P-value were indicated as followed : * : p.value <0.05, ** p.value <0.01, *** : p.value <0.001, **** p.value <0.0001.

  value <0.05) (Figure.34). However rectal enema seemed less effective than intragastric administration in our model because it does not allow to reduce the size of severe atypia (Figure.34).

group and 17715, 5

 5 µm for 3x10Gy NaCl-group) (p-value <0.05) and "atypia +" lesion (median size of 12918,5 µm for 3x10Gy FMTa and 11007,5 µm for 3x10Gy NaCl-group)(p-value <0.05) (Figure.35.A). Concerning protocols with daily administration, it seems better than the other because it only worsened lesion size for "atypia +) (median size 15133,5 µm for 3x10Gy FMTa group and 11486 µm for 3x10Gy NaCl-group) (p-value <0.05) (Figure.35.B).

Figure 34 .

 34 Figure 34. Size of the lesion according to the degree of severity in protocol 03. Lesion for the groups CTR, 3x10Gy NaCl-and 3x10Gy FMTa. P-value were indicated as followed : * : p.value <0.05, ** p.value <0.01, *** : p.value <0.001, **** p.value <0.0001.

  30) or space by several days between each (protocol 07) (Figure.31). We also decided to add the two following protocols one group receiving FMT from pooled donor faeces (Figure.30.A and

Figure 36 .

 36 Figure 36. Size of the lesion according to the degree of severity in protocol 05. A.B. Size of lesions for groups CTR, CTR NaCl-, 3x10Gy NaCl-and 3x10Gy FMTa. A. Animals from this protocol received FMTa every day from D3 to D6; time of sacrifice was on D7. B. Animals from this protocol received FMTa every day from D10 to D13 time of sacrifice was on D14. .

Figure. 31 )

 31 Figure.31).The assumption was that FMT from pooled donor faeces might improve the establishment of new microbiota in some part leading to a new start in the relationship between host and the gut microbiota.

Figure 37 .

 37 Figure 37. Size of the lesion according to the degree of severity in protocol 06. A.B.Time of sacrifice was on D14. A. Size of lesions for groups CTR, CTR NaCl-, 3x10Gy NaCl-and 3x10Gy FMTp. Animals from this protocol received FMTp every day from D0 to D3. B. Size of lesions for groups CTR, CTR NaCl-, 3x10Gy NaCl-and 3x10Gy FMTa..Animals from this protocol received FMTa every day from D0 to D3.

  ) 𝐵𝐸𝐷 = 𝐷 =1 + 𝑑 a/b > With D = nd, n the number of fractions d the dose per fraction and a/b or the number indicating radiosensitivity of concerned tissue.

Figure 40 .

 40 Figure 40. Lesion size (total and by lesion type) and immune cell recruitment. Comparison of single-dose protocols (brown for the 21Gy group, dark red for the 27Gy group) and BED equivalent fractionated dose protocols (yellow for the 3x10Gy group and red for the 3x14Gy group) over time (D14, D28 and D56).. A. Total lesion size for the 21Gy and 3x10Gy group over time in µm. B. Ulcer lesion size in the 21Gy and 3x10Gy group over time in µm. Over time, C. Atypia lesion size in the 21Gy and 3x10Gy group in µm. D. Total lesion size for the 27Gy and 3x14Gy group in µm. E. Ulcer lesion size in the 27Gy and 3x14Gy group in µm. F. Atypia lesion size in the 27Gy and 3x14Gy group in µm. G. Number of neutrophils per unit area (µm2) in 27Gy and 3x14Gy groups. H. Number of macrophages per unit area (µm2) in 27Gy and 3x14Gy groups.

Figure 41 .

 41 Figure 41. Percentage of microbial DNA sequence from sample that match microbial DNA sequence from database allowing identification of species present in samples.

Figure 42 .

 42 Figure 42. Heatmap of Bray-Curtis dissimilarity distance between two animals. A. of CTR group at D0. B.of CTR group at D56. C. of 3x10Gy group at D0. D. of 3x10Gy group at D56. E. of 3x14Gy group at D0. F. of 3x14Gy group at D56. Color from darker to brighter : the more similar to the less.

II.

  What is needed to understand the ecosystem state better?In this project, part of the aim was to explore what happens in the colic ecosystem after an exposition to ionising radiation. As mentioned earlier, studies concerning the relationship between host and gut microbiota within a context of PRD or experimental models of PRD have helped advance comprehension of symbiosis after exposure to ionising radiation but have yet to understand it completely. In addition, there are very few. What additional element bring our study?In the literature, only two studies look at gut microbiota dynamics over time in a PRD experimental model; one used brachytherapy delivered in several fractions, while the other used EBRT in a single dose[START_REF] Segers | Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation[END_REF]143). The first study by Gerrassy-Vanberg et al. examined the composition of the gut microbiota before brachytherapy (0wk) administered in three or five 1. Strength of the dynamic fractions of 5.5 or 8Gy (which is not specified) and two and six weeks (2wk and 6wk) after irradiation. No results indicating a-diversity were shown, but the authors calculated b-diversity

  module and each metabolite according to the ulceration size of each animal in the 3x14Gy group. Concerning correlation associated with microbiota alone, 298 species correlated positively or negatively with ulceration size at D56. Of these 298 species, only 86 had a change in abundance over time compared to the control group. Similarly, the correlation study between functional modules and ulceration size at D56 revealed that 321 functional modules were correlated (positively or negatively) with ulceration size at D56. Only 98 showed a difference in abundance over time compared to the control group. The 86 species and 98 functional modules were grouped according to their correlation dynamics (4 clusters) to the clinical outcome (size of the ulceration lesion at D56). Complementation studies within each cluster were performed to see if the bacteria could be associated with functional modules with the same dynamics. Of the 86 species showing changes in abundance relative to the control over time, 42 achieved completeness with 52 functional modules (out of the 98 that showed changes in abundance relative to the control over time).Most functional modules correlated with lesions that show changes in abundance relative to controls have complementarity with bacteria that show no change in abundance relative to controls. According to our study, this would mean that bacteria not modified relative to the control participate in the evolution of functional modules over time in correlation with the lesion. In conclusion, bacteria whose abundance does not change over time compared with controls could have a different phenotype in 3x14Gy compared with controls and be involved in lesions at D56.Our study of the dynamics allowed us to highlight that exposure to 3x10Gy irradiation would only appear to shift the host-microbiota mutualistic symbiosis to a transient alternative state. However, our results do not allow us to confirm this because some animals in the 3x10Gy group still show lesions and inflammation at D56. It is possible that this transient alternative state returns to a healthy stable state or switches to a stable alternative state. However, neither the microbial composition nor the metabolome results at D56 indicate that the microbiota changes.

Figure 43 .

 43 Figure 43. Percentage of host DNA proportion within the feces of animals in each group over time.

Figure 1 .

 1 Figure 1. Effect of prophylactic F. prausnitzii treatment on 29 Gy colorectal irradiation-induced histological damage to the colonic mucosa at 3 d. (a-b) Colonic sections were stained with hematoxylin, eosin and saffron (HES). (a) Sections show histological structure change of the colonic mucosa in one representative rat (4 representative sections/rat) of each group, the control group, the irradiated group and the irradiated and F. prausnitzii-treated group (Scale bars 200 µm). (b) Representative pictures showing parameters considered for histological scoring. (b1) No damage, (b2) inflammatory infiltration with a score graduation between 0 and 1 for modest inflammatory infiltration leading to slight crypt detachment from the muscularis mucosa (MM, rectangle 1) or between 1 and 2 for inflammatory infiltration observed in all the lamina propria area (rectangle 2) and (b3) epithelial damages with a score graduation between 0 and 1 for crypt morphological atypia characterized by disorganized and deformed epithelial crypts (rectangle 1) or between 1 and 2 for crypt loss (rectangle 2). Scale bars 100 µm (c) Histogram represents means±SEM of inflammatory infiltration score, epithelial damage score or total scoring including inflammatory infiltration score and epithelial damage score (for total scoring graduation of the injury was: score 0 = null; 0< score<1 = slight; 1< score<2 = moderate and score>2 = severe) quantified in control animals, in irradiated animals or in irradiated and F. prausnitzii-treated animals. Analyses were performed on 20 sections per animal and 5 animals were used per group (N = 1). Ctrl = Control, IRR = Irradiated, Fprau = F. prausnitzii. Error bars represent SEM, *p <.05, **p <.01, ***p <.001, ****p <.0001.

  to colorectal irradiation and to shed light on the mechanisms involved in the ability of F. prausnitzii to protect the colonic barrier from radiation toxicity, we assessed the mucosal profile and concentration of 17 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, IL-18, IFNγ, TNF-α, G-CSF, M, CSF and GM-CSF) using a Bio-Plex assay (Figure 4(a-b), S1). At 3 d, colorectal irradiation did not change the expression level of any measured cytokines from the colonic mucosa. Nevertheless, in rats receiving

Figure 2 .

 2 Figure 2. Effect of prophylactic F. prausnitzii treatment on 29 Gy colorectal irradiation-induced epithelial barrier breakdown at 3 d. (a-b) Measurement of ex vivo colonic trans-and paracellular permeability to HRP or FD4, respectively, using Ussing chambers. Each value represents the average of two independent measurements per animal (15-20 animals per group) and is for three independently repeated experiments (N = 3). (c) Immunohistological quantitative analysis of neutrophil infiltration in the colonic lamina propria. Colonic sections were stained by myeloperoxidase (MPO) antibody. The number of MPOpositive cells was quantified in the lamina propria and per colonic section. (d) Immunohistological quantitative analysis of resident macrophages in the colonic lamina propria. Colonic sections were stained by CD68 antibody. The area in μm 2 of CD68-positive cells stained was quantified in the lamina propria and per colonic section. For c and d, the histograms represent the average of eight independent measurements per animals (five animals per group (N = 1)). Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii. Error bars represent SEM. *p <.05, **p <.01. The graphs (e,f,g,h) represent the expression in the colonic mucosa of the chemokines GRO-KC, MIP-3α, RANTES and MCP-1 in control animals, irradiated animals or irradiated and F. prausnitzii-treated animals. ELISA multiplex assays were performed at least in duplicate per animal and 9 to 10 animals were used per group (N = 1). Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii. Error bars represent SEM, *p <.05, **p <.01.

Figure 3 .

 3 Figure 3. Microbiota composition analysis after 29 Gy colorectal irradiation in rats treated or not by F. prausnitzii. (a).16S rRNAsequencing-based alpha diversity analysis, measured by Observed, Chao1, Shannon and invSimpson index in control, irradiated and F. prausnitzii-irradiated animals. (b) Principal Coordinates of analysis of all animals, confidence ellipses appear around each group. (c) Bacteria phyla analysis in all animals. Twelve animals were used per group for these analyses (N = 1). Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii.

Figure 4 .

 4 Figure 4. Effect of prophylactic F. prausnitzii treatment on IL-1β and IL-18 production by colonic mucosal cells 3 d after 29 Gy colorectal irradiation. (a, b) Graphs representing expression in the colonic mucosa of IL-1β and IL-18 in control animals, irradiated animals, or irradiated and F. prausnitzii-treated animals. ELISA multiplex assays were performed at least in duplicate per animal and 9 to 10 animals were used per group (N = 1). Error bars represent SEM, *p <.05. (c) Representative pictures of immunofluorescence staining of colonic sections by IL-18 antibody (green) in control animals, irradiated animals (arrows show IL-18-positive immune cells) and irradiated and F. prausnitziitreated animals or by (d) IgG (negative control) in control animals. Scale bars 200 µm. Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii.

  (a-b)). The proliferative rate of crypt epithelial cells fell from 71.60 ± 0.31% of proliferative PCNA-positive cells per crypt in control rats to 64.70 ± 0.39% in irradiated rats at 3 d (p < .001). F. prausnitzii pre-treatment stabilized at basal level the proliferation of the entire array of crypt epithelial cells, including crypt stem/progenitor cells. We then measured the crypt depth, which in the colon is a criterion for mucosal thickness (Figure6(c)). At 3 d, colorectal irradiation-induced partial atrophy of the crypt (7.02% reduction of crypt depth, 202.90 ± 1.51 µm in irradiated rats vs 218.24 ± 1.58 µm in control rats, p < .001). Pretreatment of irradiated rats with F. prausnitzii maintained crypt depth at control level (220.70 ± 1.72 µm in irradiated and F. prausnitzii pre-treated rats vs 218.24 ± 1.58 µm in control rats). These results demonstrate the ability of

Figure 5 .

 5 Figure 5. Effect of prophylactic F. prausnitzii treatment on 29 Gy colorectal irradiation-induced reduction of stem/progenitor cells and tuft cells at 3 d. Representative pictures of (a) immunohistochemistry staining of colonic sections by SOX9 antibody (blue) and nuclear fast red counterstain (pink) for the quantification of crypt stem/progenitor cells (scale bars 100 µm), (b) immunofluorescence staining of colonic sections by Dclk1 antibody (in green) for the quantification of tuft cells (scale bars 100 µm), (c) immunofluorescence staining of colonic sections by IgG for negative control of DclK1 (scale bare 100 µm) and (d) immunofluorescence double-staining of colonic sections by Dclk1 and IL25 antibodies (in green and red, respectively, arrows show costaining, Scale bar 10 µm). Sections show control animals, irradiated animals, and irradiated and F. prausnitzii-treated animals for (a-b) and control animals for (c-d). Histograms representing (e) the number of SOX9-positive cells per crypt, (f) the number of Dclk1-positive tuft cells per colonic section, and (g) the number of Dclk1-and IL-25-positive tuft cells per colonic section. Histogram (e) represents the average of 50-60 independent measurements per animal (n = 5 animals per group, N = 1), histograms (f) and (g) represent the average of 8 independent measurements per animals (5 animals per group (N = 1)). Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii. Error bars represent SEM, *p <.05, **p <.01, ***p <.001.

Figure 6 .

 6 Figure 6. Effect of prophylactic F. prausnitzii treatment on 29 Gy colorectal irradiation-induced decrease of colonic mucosa renewal at 3 d and ulceration at 7 d. (a) Representative pictures of immunohistochemistry staining of colonic sections by PCNA antibody (in blue) and nuclear fast red counterstain (in pink) for the quantification of crypt epithelial proliferation in control animals, irradiated animals, and irradiated and F. prausnitziitreated animals. Scale bars 100 µm. (b) Histogram representing the percentage of proliferative PCNA-positive cells per crypt of the colon. Histograms representing quantitative analysis (c) of colonic crypt depth (µm) in each group at 3 d and (d) length of mucosal ulceration (µm) in irradiated rats and irradiated and F. prausnitzii-treated rats at 7 d, colonic sections were stained with hematoxylin, eosin and saffron (HES). For b and c, each value of the histogram represents the average of 50-60 independent measurements per animal (n = 5 animals per group, N = 1). For d, each value corresponds to the average of 20 independent measurements per animal (n = 8 animals per group, N = 1). Error bars represent SEM, **p <.01, ***p <.001 Ctrl = Controls, IRR = Irradiated, Fprau = F. prausnitzii.

  FIGURE 1. ANATOMY OF THE PELVIC AREA IN WOMEN (TOP) AND MEN (BOTTOM) (2). ..............................................................FIGURE 2. INCIDENCE RATE (Y-AXIS) FOR EACH HIGHEST INCIDENCE PELVIC AREA CANCERS (X-AXIS) (3). ........................................ FIGURE 3. STAGES OF THE LIFE CYCLE OF A CELL INCLUDING G1, S, G2 PHS (5). .......................................................................FIGURE 4. DETAILS OF THE VARIOUS STAGES OF MITOSIS (6). ................................................................................................ FIGURE 5. EXAMPLE OF A MEDICAL LINEAR ACCELERATOR USED FOR EBRT (15). .............................. ERREUR ! SIGNET NON DEFINI.FIGURE 6. EXAMPLE OF CYCLOTRON FOR MEDICAL PURPOSE (16). .........................................................................................FIGURE 7. EXAMPLES OF DOSE DISTRIBUTION FOR DIFFERENT TYPES (3D-CRT, IMRT, VMAT, PSTP, IMPT AND BT) OF RADIOTHERAPY DELIVERED IN THE CONTEXT OF PROSTATE TUMOURS (38). .................................................................... FIGURE 8. CONFIGURATION OF THE TREATMENT PLAN WITH A CT SCAN IN THE AXIAL PLANE. ...................................................... FIGURE 9. DIFFERENCE OF TUMOUR CELL DISTRIBUTION OF RADIATION BEAMS BETWEEN (A) EBRT AND (B) RADIOPHARMACEUTICAL (43). ............................................................................................................................................................. FIGURE 10.ESTIMATING THE DOSE WHERE CANCER CELLS WILL BE DAMAGED THE MOST AND NORMAL TISSUE THE LEAST (48) .........FIGURE 24. ROLE GUT MICROBIOTA IN THE FORMATION AND/OR MAINTAIN OF THE EPITHELIAL BARRIER. .................................... FIGURE 25. EXPERIMENTAL DESIGN FOR PROTOCOL 01. .................................................................................................. FIGURE 26.EXPERIMENTAL DESIGN FOR PROTOCOL 02. ...................................................................................................FIGURE 27. EXPERIMENTAL DESIGN FOR THE PROTOCOL 03. ............................................................................................FIGURE 28. EXPERIMENTAL DESIGN FOR PROTOCOL 04. ..................................................................................................FIGURE 29. EXPERIMENTAL DESIGN FOR PROTOCOL 05. ..................................................................................................FIGURE 30. EXPERIMENTAL DESIGN FOR PROTOCOL 06. ..................................................................................................FIGURE 34. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 03. ................................................... FIGURE 35.SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 04. .................................................... FIGURE 36. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 05. ................................................... FIGURE 37. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 06. ................................................... FIGURE 38. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 07. ...................................................FIGURE 39. SIZE OF THE LESION.A. TOTAL B. SEVERE FOR EACH PROTOCOLS OF FMT WITH A SACRIFICE TIME ON D14 AND CONCERNING THE GROUP 3X10GY. ...................................................................................................................FIGURE 40. LESION SIZE (TOTAL AND BY LESION TYPE) AND IMMUNE CELL RECRUITMENT. ....................................................... FIGURE 41. PERCENTAGE OF MICROBIAL DNA SEQUENCE FROM SAMPLE THAT MATCH MICROBIAL DNA SEQUENCE FROM DATABASE ALLOWING IDENTIFICATION OF SPECIES PRESENT IN SAMPLES. ..................................................................................FIGURE 42. HEATMAP OF BRAY-CURTIS DISSIMILARITY DISTANCE BETWEEN TWO ANIMALS. A. OF CTR GROUP AT D0. B.OF CTR GROUP AT D56. C. OF 3X10GY GROUP AT D0. D. OF 3X10GY GROUP AT D56. E. OF 3X14GY GROUP AT D0. F. OF 3X14GY

	Natural Treg (nTreg)
	Induced regulatory T cells (iTreg)
	Interleukin 5 (IL-5)
	Interleukin 13 (IL-13)
	Interleukin 35 (IL-35) External Beam Radiotherapy (EBRT) IL10-induced CD4 Treg (Tr1 cells) Three-Dimensional Conformal Radiation/Proton Therapy (3DCRT/3DCPT) Interleukin 17 (IL-17) Image-Guided Radiation/Proton Therapy (IGRT/IGPT Metagenomics of the Human Intestinal Tract (MetaHit) Intensive Modulated Radiation/Proton Therapy (IMRT/IMPT) Polyacrylamide gel electrophoresis (PAGE) Pencil Beams Scanning (PBS) Next generation sequencing (NGS) Helical Tomography (HT) Massively parallel sequencing (MGS) Volumetric Modulated Arc Therapy (VMAT) Pacific Bioscience (PacBio) Stereotactic irradiation (SRT) Base-pair (bp) Stereotactic Body Irradiation (SBRT) Single Molecule Real-Time (SMRT) Stereotactic Ablative Body Irradiation (SABR) Zero Mode Waveguide (ZMW) Stereotactic Body Proton Beam Therapy (SBPT) Pattern Recognition Receptor (PAR) Stereotactic Radiosurgery (SRS) Toll Like Receptor (TLR) Low-dose Rate (LDR) C-type lectin receptor (CLR) High-dose Rate (HDR) NOD-like receptor (NLR) Organs At Risk (OAR) Microbes-associated molecular pattern (MAMP) TD (tolerance dose) polysaccharides A (PSA) Dose-Volume Histogram (DVH) aryl hydrocarbon receptor (AhR) Normal tissue complication probability (NTCP) 6-formylindolo(3,2-b) carbazole (FICZ) Inflammatory bowel disease (IBD) Tight junction (TJ) Pelvic Radiation Disease (PRD) Tumor Necrosis Factor (TNFa) Common terminology criteria for adverse event (CTCAE) Interferon gamma (IFNg) Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) Colonic stem cells (CSCs) Classically Activated Macrophages (CAMµs) Antimicrobial pepides (AMPs) Alternatively Activated Macrophages (AA-Mµ) Short chain fatty acid (SCFA) Regulatry Macrophages (RMµ) Intestinal epithelial cell (IEC) Liposaccharide (LPS) Specific Pathogen Free (SPF) Toll Like Receptor (TLR) Germ-Free (GF) Interleukin 4 (IL-4) Branched-chain amino acid (BCAA) Interleukin 13 (IL-13) Bromodeoxyrudine (BrdU) Interleukin 1 (IL-1) Lipopolysaccharide (LPS) Interluekin 10 (IL-10) Nitric oxyde (NO) Interleukin 6 (IL-6) Prostaglandin E2 (PGE2) Denditric Cell (DC) Reactive Oxygen Species (ROS) Interleukin 12 (IL-12) Taurocholic Acid (TLC) Interleukin 18(IL-18) Myeloid differentiation factor 88 (MyD88) Interleukin 15(IL-15) Radiation enteritis (RE) Interleukin 2(IL-2) Trans-Epithelial Electrical Resistance (TEER) Natural Killer (NK) World Organism of Health (WHO) Natural Killer Cell (NKc) European Food Safety Authority (Efsa) Innate Lymphoid Cell (ILC) Faecal Microbiota Transfer (FMT) Regulatory B cells (Bregs) Biologically Effective Dose (BED) Cytotoxic (LTCD8 or LT killer) radiation-induced gastrointestinal syndrome (RIGS) Non cytotoxic (LT helper (LTh), LTCD4) Total Body Irradiation (TBI) T-Regulator (LTreg) Mucosal-Associated Invariant T cells (MAIT) Dextran Sulfate Sodium (DSS)
	Lymphocyte dg (LTdg)
	T-cell receptor (TCR)

FIGURE 11. EXPLANATION OF GRADE OF CTCAE FOR DIARRHEA AND COLITIS (LEFT) AND RTOG (RIGHT) CONCERNING TOXICITY TO THE BOWEL RELATED TO ANTI-CANCER DRUG AND IRRADIATION (61,62). .................................................................... FIGURE 12. STRUCTURAL ORGANISATION OF THE LARGE INTESTINE. DETAILS OF THE DIFFERENT LAYERS OF THE COLONIC TISSUE (66). ....................................................................................................................................................................

FIGURE 13. DIFFERENTIATED COLONIC EPITHELIAL CELLS. ...................................................................................................

FIGURE 14. DESCRIPTION OF THE FUNCTION OF MACROPHAGES ACCORDING TO TYPE AND SUBTYPE AND WHICH MEDIATOR PROMOTES WHICH PHENOTYPE. .......................................................................................................................................... FIGURE 15. ALL SUBTYPE OF ILC REUNITING NKC ILC1, ILC2, ILC3 AND LTI AND THEIR ROLES. ................................................ FIGURE 16. DESCRIPTION OF ALL SUBTYPES OF LT AND LB THUS THEIR DIFFERENT ACTIONS ...................................................... FIGURE 17. THE MUCOSA OF THE COLON IN NORMAL CONDITION WITH ITS DIFFERENT SECTIONS. .............................................. FIGURE 18. DETAILED EVENT COULD HAPPEN ON TISSUE AFTER EXPOSURE TO IONIZING RADIATION (43). .................................... FIGURE 19. EXAMPLE OF DIFFERENT SYMBIOSIS TYPE. .......................................................................................................

FIGURE 20. STAGES OF SANGER SEQUENCING METHODS ................................................................................................... FIGURE 21. SECOND-GENERATION OF SEQUENCER. .......................................................................................................... FIGURE 22. OPERATION OF THIRD GENERATION SEQUENCERS. ............................................................................................

FIGURE 23. EXAMPLE OF A PHYLOGENIC TREE. ................................................................................................................. FIGURE 31. EXPERIMENTAL DESIGN FOR PROTOCOL 07. ..................................................................................................

FIGURE 32. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 01. ...................................................

FIGURE 33. SIZE OF THE LESION ACCORDING TO THE DEGREE OF SEVERITY IN PROTOCOL 02. ................................................... GROUP AT D56. COLOR FROM DARKER TO BRIGHTER : THE MORE SIMILAR TO THE LESS. ...............................................

FIGURE 43. PERCENTAGE OF HOST DNA PROPORTION WITHIN THE FECES OF ANIMALS IN EACH GROUP OVER TIME. ....................

TABLE 1. MOST COMMON SEALED RADIOACTIVE SOURCE IN BRACHYTHERAPY FOR PELVIC CANCER (38) ERREUR ! SIGNET NON DEFINI.

TABLE 2

 2

. REVIEW OF LITERATURE CONCERNING DOSE CONSTRAINT TO LIMITED RADIATION-INDUCED TOXICITY FOR TREATED CANCER IN THE PELVIC AREA. ORGAN AT RISK IDENTIFIED INCLUDE BULB OF PENIS, ANAL CANAL, COLON/SIGMOID, SMALL BOWEL, BONE MARROW, UTERUS, BLADDER, ILIAC BONE, OVARY, LUMBOSACRAL PLEXUS, RECTUM, SACRUM, FEMORAL HEAD (50). .......... TABLE 3. DESCRIPTION OF THE DIFFERENT RELATIONSHIP EXISTING BETWEEN LIVING ORGANISMS. .............................................. TABLE 4. RESUME OF TECHNIQUES, ADVANTAGE AND INCONVENIENCE OF EACH SEQUENCING PLATFORM.. .................................. TABLE 5. EXAMPLE OF THE CLASSIFICATION OF THE BACTERIA AKKERMANSIA MUCINIPHILA WITH THE NAMES OF IT DOMAIN, KINGDOM PHYLUM, CLASS, ORDER, FAMILY, GENUS, SPECIES AND STRAIN. .................................................................................

TABLE 6 .

 6 GUIDELINES CONCERNING SOME OF THE TREATMENT PATIENT DECLARING AFTER PELVIC RADIOTHERAPY. AMONG THEM GUIDELINES FOR DIARRHEA, CONSTIPATION, FECAL INCONTINENCE, RECTAL BLEEDING (139). ..........................................

TABLE 7 .

 7 SUMMARY OF PROTOCOLS USED IN FOUR EXPERIMENTAL STUDIES OF PROBIOTICS IN PRD. ...........................................

Table 2 .

 2 Review

of literature concerning dose constraint to limited radiation-induced toxicity for treated cancer in the pelvic area. Organ at risk identified include bulb of penis, anal canal, colon/sigmoid, small bowel, bone marrow, uterus, bladder, iliac bone, ovary, lumbosacral plexus, rectum, sacrum, femoral head

Table 3 .

 3 Description of the different relationship existing between living organisms.

	Nature of relation	Species A	Species B
	Parasitism	-	+
	Mutualism	+	+
	Commensalism	0	+
	Competition	-	-
	Amensalism	0	-
	Neutralism	0	0

Table 4 .

 4 Resume of techniques, advantage and inconvenience of each sequencing platform.When sequencing a microbiota, different options are available. It is possible to sequence only a targeted region of the DNA that can differentiate between the species in the microbiota or to sequence the entire genome for all molecules detected in a sample. As mentioned earlier, sequencing a specific portion of the DNA could target an amplicon such as the gene encoding 16SrRNA. Both techniques have advantages and disadvantages, and the choice of sequencing technique again depends on the question.

	Plateform Technique	Advantage	Inconvenient
	Sanger	Fluorescence	Long read	Time-consuming
				Expensive
	Roche	Pyrosequencing Long read	Time-consuming
				Need	PCR	(create
				biases)
				Not used since 2016
	Illumina Fluorescence	Hight accuracy	Time-consuming
			Hight throughput	Need PCR
				Short read
	Ion	Proton	Medium read	Need PCR
			Rapid	Length of nucleotide
				repetition hard to detect
				Low throughput
	PacBio	Fluorescence	Long read	Time-consuming
			Easily detecting complex genomic	Expensive
			region	Low throughput
			Hight accuracy
	Oxford	Electronic	Long read	Hight error rate
	Nanopore	signals	Rapid	Low throughput
			Easily detecting complex genomic
			region

Table 5 .

 5 Example

of the classification of the bacteria Akkermansia muciniphila with the names of it domain, kingdom phylum, class, order, family, genus, species and strain.

Table 7 .

 7 Summary of protocols used in four experimental studies of probiotics in PRD.

Grad es 500µm B Grade 1 Grade 2 Grade 3 Mucosal crypt damage score

	All
	A delimited by the
	rectangle, Ba,Ca for 3X10Gy D14, Bb,Cb for 3X10Gy D28, Bc,Cc for 3X10Gy D56, Bd,Cd for 3X14Gy
	D14, Be,Ce for 3X14Gy D56 and Bf,Cf for 3X14Gy D56. Boxplots represent the median (+ upper
	quartile (Q3, 75% of the data)) / (-lower quartile (Q1, 25% of the data)) of the length of D total lesions,
	E grade 1 lesions, F grade 2 lesions and G grade 3 lesions. H Boxplots represent the median (+Q3/-
	Q1) of the mucosal crypt damages scores calculated considering each grade's proportion (length)

within the total lesion, normalised to the maximal lesion length measured after irradiation. Analyses were performed on 5 sections per animal and 6 rats/group were used at D14 and D28 and

12

at D56 (N=1). Significances between group (*) and time (letter) were calculated with Student, Welch or Mann-Whitney test according to normality and variance equality* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Boxplots sharing the same letter and colour letters are not different.

Table .

 .

	S3 and Supplementary Table.S4). Using a mathematical tool
	(clustering of time-course data) for group species with similar dynamics, we reported 6
	temporal profiles after 3X10Gy and 3X14Gy irradiation (Fig 4B-D). Profiles ranged from
	transient increase or decrease of the abundance of bacteria species either in the early period
	(cluster 1_sp and 3_sp for 3X10Gy and Cluster 6_sp for 3X14Gy, respectively), medium period
	(cluster 2_sp for 3X10Gy) or in a period spanning the entire measured time scale (cluster 4_sp

  5 different clusters for moderate and 6 for severe lesions (Fig 9B, Supplementary Table. S10 and Table. S11). We selected 5 clusters (F_met, E_met, H_met, J_met and K_met) in which, at least, one of the glutamate, arginine, ornithine or proline metabolites is represented. Clusters F_met and E_met for which positive correlations with moderate lesion increase respectively gradually over time or only for the latest times (D40 to D56) include the ornithine, glutamate and proline for the first one and only

arginine for the second. Concerning severe lesions, glutamate belongs to cluster K_met showing a gradual positive correlation over time, ornithine and proline to respectively H_met

an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy. Nucleic Acids Research. 2022 Jan

Acknowledgments:

The authors wish to thank GSEA of the IRSN for the technical assistance provided with the animals.

Acknowledgments

The authors wish to thank GSEA of the IRSN for the technical assistance provided with the animals. This work has benefited from the facilities and expertise of @BRIDGe (GABI, INRA, AgroParisTech, Paris-Saclay University.

(145) Table 6. Guidelines concerning some of the treatment patient declaring after pelvic radiotherapy. Among them guidelines for diarrhea, constipation, fecal incontinence, rectal bleeding (139).

THESIS AIMS SCIENTIFIC PAPER AND SUPPLEMENTARY RESULTS

Conflicts of interest:

The authors declare that there is no conflicts of interest regarding the publication of this article

Author contribution:

MG: Design of the work, mathematical and statistical analyses, data analysis and interpretation, revision of the drafting of the manuscript, OGQ: Data analysis, mathematical and statistical analyses, MAB: Mathematical and statistical analyses, revision of the drafting of the manuscript, MS: Interpretation of metabolomic data, revision of the drafting of the manuscript, Stéphane Grison: Data analysis, interpretation of metabolomic data, revision of the drafting of the manuscript, JCM: Interpretation of metabolomic data, revision of the drafting of the manuscript, JMC: Data interpretation, revision of the drafting of the manuscript, JD: Data interpretation, revision of the drafting of the manuscript, MA: Mathematical analyses, data acquisition, interpretation of metagenomic data, revision of the drafting of the manuscript, FM: Conception and design of the work, data interpretation, have drafted the work and , AS:. Conception and design of the work, data interpretation and have drafted the work.

SUPPLEMENTARY FIGURES S1 TO S7 AND TABLES S1 TO S11

Supplementary Figure S1: Geiger M et al.

Supplementary Fig S1:

Experimental protocols 1 and 2 were used for histological analysis, lesion severity and immune recruitment (Protocol 1 and D56 in Protocol 2) and for the study of microbial and metabolite composition after irradiation, followed by analysis of the correlation between the presence of grade 2 and grade 3 lesions at D56 with the evolution of the microbial and metabolite composition (Protocol 2). Supplementary Table S1 : List of phyla, order, family and genus significantly modulated in the 3X10 Gy group vs control (CTR) group using linear regression model . Cluster annotation for each variable is shown.

Supplementary Table S1

Supplementary Table S2 : List of phyla, order, family and genus significantly modulated in the 3X14 Gy group vs control (CTR) group using linear regression model. Cluster annotation for each variable is shown..

Supplementary Table S2

Supplementary Table S3: List of 66 species significantly modulated in the 3x10Gy group vs control (CTR) group using the lineat regression model. Cluster annotation for each variable is shown

Supplementary Table S3 Supplementary Table S4 Supplementary Table S4 Supplementary Table S8 : List of the 289 functional modules in the 3X14 Gy group which dynamic overtime correlates with a moderate lesion (grade 2) at D56.

Supplementary Table S8

Supplementary Table S9 : List of the 301 functional modules in the 3X14 Gy group which dynamic overtime correlates with a severe lesion (grade 3) at D56.

Supplementary Table S9

Supplementary Table S10 : List of the 194 metabolites in the 3X14 Gy group which dynamic overtime correlates with a moderate lesion (grade 2) at D56.

Supplementary table S10

Supplementary Table S11 : List of the 138 metabolites in the 3X14 Gy group which dynamic overtime correlates with a severe lesion (grade 3) at D56.

Supplementary Table S11

Whereas we thought having a great and consistent administration protocol these results establish that it was not the case.

We aimed to see if FMT could be interesting in preventive and curative treatment. Because the preventive protocol seems inconsistent, we wanted to try the same administration protocol in the curative one, where we began to administrate treatment days after the last dose of irradiation.

At this time of the project, we just received our shotgun sequencing results, and we could refer to them to try protocol according to how gut microbiota evolve. We decided to establish one protocol with a beginning of administration on D3 because it was the time when we identified that microbiota was evolving (Figure .29.A) (richness). We also tried protocol with a beginning of treatment on D10 because we thought that in an ecosystem with fewer dying cell messages (because after the apoptosis phase), less severe lesion microbiota from transfer could be better implanted (Figure .29B).

Both protocols from 05 one showed amelioration in the lesion size after treatment with FMTa with better improvement for protocol 05B. Effectively for protocol 05A, reduction of "atypia +" lesion size was observed in the 3x10Gy FMTa group (median size of 2396,69 µm for 3x10Gy FMTa group and 3707,055 µm for 3x10Gy NaCl-group) (p-value <0.05). For protocol 05B "atypia+" lesion size was not reduced. However, three other types of lesion were: the A B It is also possible that the lesions and the toxic environment are still too important for FMT to work, even if this has been minimised.

FMT as a preventive treatment for radiation-induced colonic lesions does not seem effective unless other stresses impacting the microbiota and/or the host occur, regardless of the protocol.

We decided to test another protocol which was this time curative.

To see if FMT could be interesting as a curative treatment, we designed a protocol in which we treated the animals ten days after the last irradiation fraction in the 3x10Gy protocol. At this stage, the gut microbiota appears identical to the control's, but the host shows inflammation and lesions. We cannot confirm to what extent the damage to the host is greater or less than before (i.e., from the day of the last irradiation fraction to D07-D14) because we do not have a sample before D07 for lesions and D14 for inflammation. Nevertheless, we believe host lesions are more important at D07 than D14. Either way, in this context, we found an effect of FMT.

We did not replicate this experiment, so we cannot say this effect is constant. Nevertheless, we never have as much effect in our preventive treatment when the animals are unaffected by any other stress. FMT may have a positive effect only when the environment is not too hostile for the gut microbiota from FMT which would imply it is the case at D10 compared to D0 in the 3x10Gy irradiation protocol. For these reasons, it might be interesting to replicate this In this study, we had two objectives regarding the analysis of the gut microbiota: to estimate the diversity and establish a functional profile according to the microbial composition. It is possible to opt for 16S rRNA analysis or shotgun analysis to analyse the gut microbiota composition. Regarding diversity assessment, one practice has no advantage over the other except for practical reasons. 16S rRNA is cheaper, allowing more analysis time points and/or animals. It is also less difficult to analyse, so it seems to be the best solution if the sole purpose is to know the microbial diversity. Identifying the diversity of the microbial composition in each sample is part of what we are looking for. It will allow us to see if there is a microbial signature between rats exposed to irradiation and, if so, if this signature can be correlated with host lesions. But we want to go further and understand the consequences of the presence of a species in an ecosystem. It is possible to predict the functions of species in each microbiota through DNA sequence. As mentioned, tools to predict function built for 16SrRNA are not optimal. It is the advantages of shotgun sequencing that led us to choose it.

B. Curative treatment

Furthermore, we performed faecal metabolomics measurements on the same animals in parallel with the faecal microbiota analysis. With metabolomic data, it is difficult to differentiate between metabolites from the host and those from the microbiota. The advantage of having a tool that predicts the functional module in which microorganisms are involved is to crossreference the information between the predictive and metabolomic data to identify metabolites from the microbiota. The prediction of the functional module we are looking for must be as reliable as possible and contain the greatest diversity of functionality to increase our chances of correlating with our metabolomic study; therefore, we choose shotgun over 16S rRNA sequencing. These three types of analysis would make it possible to fully answer the question of which species populate the intestinal microbiota, what they do and what the consequences are for the relationship with the host.

We are interested in the evolution of the relationship between the host and the microbiota in the colonic ecosystem over time. Therefore, the most interesting option was to carry out a

Design for gut microbiota analysis

A. Type of analysis B. Longitudinal study from the radiation-resistant mice to control mice, which they then exposed to irradiation. These mice, which after FMT had a microbial signature in common with their donor, were better able to withstand irradiation than mice that had received a different microbiota Moreover, authors, performed metabolomic studies and found metabolites of interest enriched in the radiationresistant group. Administration of one of these metabolites (propionate) prior to irradiation increased the survival rate of the animals after irradiation. According to the authors, this radiation protection from propionate administration may be related to the fact that propionatetreated animals had much lower ROS levels compared to those that did not receive propionate.

This study highlights that the damage created by irradiation may be related to the microbiota, like the side effects of anticancer drugs. However, this study was performed in mice without tumours and therefore did not evaluate the therapeutic efficacy in mice developing fewer side effects. It would be interesting, as with chemotherapy and immunotherapy, to continue the study of radiation-induced effects and microbiota in a tumour context. This could allow us to identify whether there may be microbiota, or metabolites derived from this microbiota, which could allow patients to avoid the development of side effects without reducing the anti-cancer effect of their treatment.

In conclusion, FMT appears to be a therapeutic option for several pathologies that could be used at different points in the therapeutic process. However, research into understanding the function of the ecosystem needs to progress to optimise the therapeutic capacity of FMT. In addition, much work must be done on preparing the transfer to increase the chances of implantation and, therefore, a therapeutic response in patients.

GENERAL CONCLUSION

ANNEXE

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kgmi20 Gray in the colon resulting in colonic epithelial barrier rupture. Three days before the irradiation and up to 3 d after the irradiation, the F. prausnitzii A2-165 strain was administered daily (intragastrically) to test its putative protective effects. Results showed that prophylactic F. prausnitzii treatment limits radiation-induced para-cellular hyperpermeability, as well as the infiltration of neutrophils (MPO+ cells) in the colonic mucosa. Moreover, F. prausnitzii treatment reduced the severity of the morphological change of crypts, but also preserved the pool of Sox-9+ stem/progenitor cells, the proliferating epithelial PCNA+ crypt cells and the Dclk1+/IL-25+ differentiated epithelial tuft cells. The benefit of F. prausnitzii was associated with increased production of IL-18 by colonic crypt epithelial cells. Thus, F. prausnitzii treatment protected the epithelial colonic barrier from colorectal irradiation. Newgeneration probiotics may be promising prophylactic treatments to reduce acute side effects in patients treated with radiation therapy and may improve their quality of life.

Ethics statement

All experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animal as published by the French regulations for animal experiments (Ministry of Agriculture Order No. B92-032-01, 2006) and with European Directives (86/609/CEE) and were approved by the local ethics committee of our institute (permit number P16-07).

Animals

Male SD (Sprague Dawley) rats (Janvier SA, Le Genest St Isle, France) weighing 250-300 g were

Competing financial interest declaration

The authors declare no conflicts of interest.