Treatment or prevention of gastrointestinal barrier dysfunction

Abstract

Herein are provided compositions comprising lysate of the bacterial species Methylococcus capsulates (Bath) for use in the treatment of chemotherapy-induced bowel injury, antibiotics-induced diarrhoea and radiation-induced bowel injury. Also provided are methods of treatment comprising administering said composition, as well as kits comprising said composition.

Description

[0001] P7524EP00

[0002] Treatment or prevention of gastrointestinal barrier dysfunction

[0003] Technical field

[0004] The present invention relates to compositions comprising lysate of the soil bacterium Methylococcus capsulates (Bath) for use in treatment or prevention of gastrointestinal barrier dysfunction, such as chemotherapy- or radiation-induced bowel injury in the small or large intestine, or antibiotics-induced diarrhoea.

[0005] Background

[0006] With the increasing global population, food demands are increasing. Traditional means of food production heavily deplete natural resources, cause profound emission of greenhouse gasses, and contribute to the deteriorating global biodiversity. Particularly whether protein supply can match demands raise concern. To accommodate these needs, alternative protein sources, such as insect- or microbe-based proteins, may serve as a sustainable solution to the decline in natural resources.

[0007] Dietary proteins are not only nutritional building blocks but may also contribute to modulation of immune regulation, as demonstrated by their roles in vaccine design and food allergies. Besides influencing gut microbial composition, protein-derived peptides presented on major histocompatibility complex (MHC) molecules activate mucosal T cells, which play a central role in maintaining gastrointestinal (Gl) immune homeostasis. Among these, peripherally induced regulatory T-cells (pTregs) are critical for suppressing aberrant immune responses towards food antigens and innocuous microbes.

[0008] T-helper 17 (Th17) cells are another important cell type regulated by microbial signals. Through production of interleukin 17 (IL-17) they demonstrate pleiotropic effects, fortifying epithelial barriers under homeostatic conditions while fueling inflammation in inflammatory bowel disease (IBD). Thus, depending on type and duration of antigen stimulation and cytokine exposures, Th17 cells may take on immunosuppressive, barrier-strengthening or proinflammatory profiles. High levels of transforming growth factor p (TGF-p) and IL-27 can promote expression of anti-inflammatory cytokines, including IL-10, alongside IL-17, whereas I L-1 p can promote transdifferentiation to T-helper 1 (Thl)-like states, allowing for expression of pro-inflammatory interferon y (I FN-Y).P7524EP00

[0009] Disturbances in the balance between immune activation and tolerance at the Gl interface can exacerbate gastrointestinal barrier dysfunction. Initial gastrointestinal barrier dysfunction may arise as a result of several different causes, e.g. as a result of the cytotoxic effects of chemotherapeutic agents on rapidly dividing cells in the gastrointestinal tract, as a result of disruption of the normal gut microbiota balance by antibiotics, particularly broad-spectrum antibiotics, or due to the exposure of intestinal tissues to ionizing radiation e.g. during cancer treatment.

[0010] However, current interventions targeting host-microbe interactions, including probiotics and fecal transplants, often yield inconsistent results due to the context-dependent behavior of microbes.

[0011] There is thus an unmet need for compositions that can reliably restore gastrointestinal barrier dysfunctions, and preferably wherein said compositions can simultaneously provide an alternative source of protein.

[0012] Summary

[0013] The inventors have surprisingly found that lysate from the non-native bacterium, Methylococcus capsulatus Bath (McB), which is a fully scalable and sustainable, protein source, and is already characterized and commercially available, has immunomodulatory and regenerative properties on the gastrointestinal barrier. Indeed, this McB-based lysate rapidly alters the gut microbiota and regulates mucosal T cells in a diet independent, spatiotemporal manner. The lysate induces pTreg expansion independently of the gut microbiota, may induce 10+Th17 cells, and has regenerative potential in gastrointestinal (Gl) inflammation. These properties have particular benefits for treating chemotherapy- and radiation-induced mucositis as well as antibiotics-induced diarrhoea, which are often characterized by a severely compromised gut barrier function.

[0014] Chemotherapy- and radiation-induced injury target rapidly dividing cells, making these treatments essential to combat various fast-growing cancers. However, the intestinal epithelium is similarly rapidly dividing to counter the contents exposures to exogenous stimuli from the lumen, with a complete turnover of the entire gut lining within a few days. Thus, patients undergoing chemotherapy and / or radiation therapy often developP7524EP00

[0015] intestinal inflammation as a notable side-effect. This is very well-described for colorectal cancer patients treated with the chemotherapy 5-fluorouracil (5-Fll). As a result of the damage to the intestinal epithelium, small intestinal villi and intestinal crypts deteriorate, causing villus atrophy, crypt shortening, and reduced colon length.

[0016] The inventors have observed that McB-based lysates protect against 5-FU-induced gut barrier dysfunctions, effectively alleviating villus atrophy and crypt shortening, with preserved colon function. As another testament to the gut-protective effects of McB-based lysates, the investors have demonstrated that the compositions of the present disclosure mitigate gut barrier dysfunction caused by antibiotics-exposure. Antibiotics are known to impact the intestinal lining in multiple ways, including indirectly by reducing gut microbial load, thus removing stimuli from beneficial, barrier-strengthening microbial signals, in addition to the fermentative functions of the gut microbiota.

[0017] Moreover, antibiotic exposure causes profound intestinal cellular stress, reducing mucus production and mucus integrity, thereby facilitating bacterial encroachment and proinflammatory reactions in the gut. Combined, these factors contribute to the often-accompanied antibiotics-induced diarrhea and even weight loss. The inventors have found that, surprisingly, the presently disclosed compositions also protect against antibiotics-induced diarrhea and weight loss. Additionally, the inventors have observed that the prophylactic, ameliorating and / or curative effects does not seem to be a result of increased secretion of intestinotrophic gut hormones, but, surprisingly, may be mediated by fermentation by the native gut microbiota in order to confer its gut-protective effects.

[0018] The etiologies of chemotherapy- or radiation-induced bowel injury, or antibiotics-induced diarrhoea, differ substantially from the previously described gut barrier dysfunctions described as responsive to treatment with McB, namely obesity / metabolic-induced gut dysfunction (Jensen et al., 2021) and chemically induced colitis (Kleiveland et al., 2013). Obesity-induced gut barrier damage is mainly caused by weakening of tight junctions between epithelial cells and reduced mucus production, slightly increasing contact with the gut microbiota and dissemination of bacterial products, causing local and systemic low-grade inflammation. Chemically induced colitis is primarily caused by direct cytotoxic effects, effectively destroying the intestinal epithelial layer, aggravated by microbial infiltration and subsequent inflammation.P7524EP00

[0019] It is therefore surprising that the present compositions provide prophylactic, ameliorating and / or curative effects, such as directly strengthen the gut barrier, prevent atrophy of the epithelial layer, and restore the integrity of the mucosae, in the above-mentioned gut barrier dysfunctions that differ substantially from obesity / metabolic-induced gut dysfunction and chemically induced colitis.

[0020] The present disclosure therefore provides nutritional compositions with therapeutic potential to alleviate Gl inflammation. As shown in the examples of the present disclosure, compositions comprising the lysate of the present disclosure may reduce gastrointestinal barrier dysfunction severity, mitigated gastrointestinal barrier dysfunction-induced villus destruction and restore mucosal integrity.

[0021] It is an aspect of the present disclosure to provide a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of:

[0022] a. chemotherapy-induced bowel injury;

[0023] b. antibiotics-induced diarrhoea; or

[0024] c. radiation-induced bowel injury.

[0025] Also provided is a method of treatment or prevention of

[0026] a. chemotherapy-induced bowel injury;

[0027] b. antibiotics-induced diarrhoea; or

[0028] c. radiation-induced bowel injury,

[0029] said method comprising administering to a subject in need thereof an effective amount of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath).

[0030] Additionally provided is the use of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for the manufacture of a medicament for treatment of:

[0031] a. chemotherapy-induced bowel injury;

[0032] b. antibiotics-induced diarrhoea; or

[0033] c. radiation-induced bowel injury.

[0034] In another aspect is provided a kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating chemotherapy-P7524EP00

[0035] induced bowel injury, antibiotics-induced diarrhea, and / or radiation-induced bowel injury.

[0036] Description of Figures

[0037] Figure 1. McB lysates rapidly imprints the gut microbiota and mucosal immune landscape. (A) Study design. (B) Differentially abundant taxa associated with McB based on a general mixed linear model with subject as a random variable. (C) Relative abundance levels of Methylococcus capsulatus Bath across diets and time. (D) Relative abundance plots of top 12 species differentially regulated by McB as determined by general mixed linear modelling. (E) Bubble plot of top 10 significantly regulated microbial metabolic pathways differentiating reference diets and McB-containing diets. Size of each dot indicated the center log-ration transformed pathway abundance as determined by ALDEx2. (F-l) Flow cytometry analysis of small and large intestinal lamina propria T-cells in Simple Diet-fed mice, showing levels of FoxP3+RORyt+peripherally induced regulatory T-cells (pTregs) in small intestine (F) and large intestine (G), FoxP3'RORyt+T-helper 17 (Th17) cells in small intestines (H) and large intestines (I). (J-M) Flow cytometry analysis of small and large intestinal lamina propria T-cells in Complex Diet-fed mice, showing levels of FoxP3+RORyt+peripherally induced regulatory T-cells (pTregs) in small intestine (J) and large intestine (K), FoxP3'RORyt+T-helper 17 (Th17) cell in small intestines (L) and large intestines (M). (F-M) Data are presented as mean ± SEM (n = 3-6 per group) with individual data points. Statistical significance between two groups was determined by unpaired t-test (P < 0.05).

[0038] Figure 2. McB-mediated immune imprinting is subset specific. (A) Study design. (B) Relative cecum sizes over time in response to antibiotics (ABX) treatment. (C, D) Levels of FoxP3+RORyt+peripherally induced regulatory T-cells (pTregs) over 2, 4, and 6 weeks in small intestine (C) and large intestine (D) lamina propria. (E-G) Levels of FoxP3'RORyt+T-helper 17 (Th17) cells in small intestinal lamina propria after 2 (E), 4 (F), and 6 weeks (G). (H-J) Levels of FoxP3'RORyt+Th17 cells in large intestinal lamina propria after 2 (H), 4 (I), and 6 weeks (J). Data are presented as mean ± SEM (n = 3-4 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test. P < 0.05 was considered statistically significant.P7524EP00

[0039] Figure 3. McB consumption protects against gastrointestinal injury. (A) Small intestinal mucositis study design. (B) Body weight development post injection with 5-fluorouracil (5-FU) (400 mg / kg) as relative to baseline weight of acute - day 1-3 - and recovery phases - day 4-6. (C) Area under the curve analysis of body weight loss following 5-FU injection. (D) Small intestinal wet weights in grams during acute phase (Day 3) and recovery phase (Day 6). (E) Jejunum villus length in pm during acute phase (Day 3) and recovery phase (Day 6). (F) Ileum villus length in pm during acute phase (Day 3) and recovery phase (Day 6). (G) Large intestinal length in cm during acute phase (Day 3) and recovery phase (Day 6). (H) HE images of jejunum and ileum villi, representing the median within each group. Scalebars = 50 pm. Scale bars = 100 pm. Data are presented as mean ± SEM (n = 9 per group in mucositis protocol) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test (C-G). P < 0.05 was considered statistically significant.

[0040] Figure 4. McB-mediated small intestinal protection is GLP-2R dependent. (A) Study design of beginning of recovery phase after induction of small intestinal mucositis using the 5-fluorouracil model in whole-body GLP-2R wildtype (WT) and knockout (KO) mice. (B-G) Body weight development following small intestinal injury (B), small intestinal wet weights (C), Jejunum (D) and ileum (E) villus lengths, and jejunum (F) and ileum (G) myeloperoxidase levels corrected for total protein content, in WT mice. (H-M) Body weight development following small intestinal injury (H), small intestinal wet weights (I), Jejunum (J) and ileum (K) villus lengths, and jejunum (L) and ileum (M) myeloperoxidase levels corrected for total protein content, in KO mice. (N) Histological images of jejunum and ileum sections of WT and KO mice, representing group medians. Scalebars = 50 pm. Data are presented as mean ± SEM (n = 6-10 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test. P < 0.05 was considered statistically significant.

[0041] Figure 5. Acute and recovered duodenum villus lengths in the 5-fluorouracil mucositis model. Data are presented as mean ± SEM (n = 7-9 per group) with individual data points. Statistical significance was determined by Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test. P < 0.05 was considered statisticallyP7524EP00

[0042] significant. Grey bars indicate Complex-ref Diet fed mice; green bars indicate Complex-McB fed mice.

[0043] Figure 6. (A, B) Duodenum villus lengths in GLP-2R wildtype (WT) (A) and knockout (KO) mice (B). Data are presented as mean ± SEM (n = 7-10 per group) with individual data points. Statistical significance was determined by Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test. P < 0.05 was considered statistically significant.

[0044] Figure 7. A) Schematic of the perfused rat intestine model, with venous effluent collected every minute from the portal vein. (B) GLP-1 levels in venous effluent for 90-minutes perfusion, with shaded areas indicating periods of stimulation. GLP-1 was used as a proxy for GLP-2 as they are co-released by L-cells. (C) Levels of absorbed amino acids in vascular effluent across 80 minutes with shaded areas indicating periods of stimulation. (D) Schematic of mouse gavage study. (E) Plasma levels of GLP-2 following oral gavage of McB, reference, or a control glucose solution at 200 mg / mL in PBS. Mice and rats were given an inhibitor cocktail consisting of sacubitril (neprilysin inhibitor, 0.3 mg / kg, 5 uL / g) and sitagliptin (dipeptidyl peptidase-4 inhibitor, 10 mg / kg, 5 uL / g) 30 minutes before oral delivery of solutions, n = 4 Sprague Dawley rats per group, and n = 3-6 mice per group, as indicated.

[0045] Figure 8. (A) Study design of the dextran sodium sulphate (DSS) colitis model protocol. Mice were given control drinking water or a 20-ppm solution of an inhibitor of microbial hindgut fermentation (Fermlnh). (B-G) Disease assessment in reference diet-fed mice. (B) Disease activity index (DAI) over the entire study period. (C) DAI at termination. (D) Colon weight-to-length inflammation index. (E) Colon length in cm. (F) Small intestine lengths in cm. (G) Histopathological scoring of colitis assessed blinded in refence diet-fed mice. (H-M) Disease assessment in McB-fed mice. (H) DAI over the entire study period. (I) DAI at termination. (J) Colon weight-to-length inflammation index. (K) Colon length in cm. (L) Small intestine lengths in cm. (M) Histopathological scoring of colitis assessed blinded in McB diet-fed mice. (N) GLP-2 levels in plasma of mice fed the reference diet or McB-diet with and without Fermlnh. Data are presented as mean ± SEM with individual data points for each mouse (n = 10-12 / group).

[0046] Statistical significance was determined by a Two-Way ANOVA followed by Fisher’sP7524EP00

[0047] LSD multiple comparisons post hoc test. P < 0.05 was considered statistically significant.

[0048] Figure 9. McB-mediated production of peripherally induced regulatory T-cells (pTregs) is not dependent on microbial fermentation. The figure shows levels of FoxP3+RORyt+pTregs from small intestine. Data are presented as mean ± SEM (n = 8-9 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Tukey’s multiple comparisons post hoc test. P < 0.05 was considered statistically significant.

[0049] Detailed description

[0050] Definitions

[0051] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.

[0052] The term “Methylococcus capsulates (Bath)” refers to a specific strain (Bath) of the obligately methanotrophic gram-negative, non-motile coccoid bacterium species Methylococcus capsulates. In some embodiments, Methylococcus capsulates (Bath) refers to the bacterial strain deposited in the National Collection of Industrial, Food and Marine Bacteria with the accession number NCIMB 11132.

[0053] The term "therapeutically effective amount" of a compound as used herein refers to an amount sufficient to cure, alleviate, prevent, reduce the risk of, or partially arrest the clinical manifestations of a given disease or disorder and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the individual. It will be understood that determining an appropriate dosage may be achieved e.g. using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary. The term “an effective amount” is used interchangeably with the term “therapeutically effective amount" unless otherwise specified.

[0054] The terms “treatment” and “treating” as used herein refer to the management and care of an individual for the purpose of combating a condition, disease or disorder. The termP7524EP00

[0055] is intended to include the full spectrum of treatments for a given condition from which the individual is suffering. The individual to be treated is preferably a mammal, in particular a human being. Treatment of animals, such as mice, rats, dogs, cats, horses, cows, sheep and pigs, is, however, also within the scope of the present context. The individuals to be treated can be of various ages.

[0056] Methods of treatment

[0057] In some aspects of the present disclosure is provided a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of:

[0058] a. chemotherapy-induced bowel injury;

[0059] b. antibiotics-induced diarrhoea; or

[0060] c. radiation-induced bowel injury.

[0061] Thus, in some embodiments is provided a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of chemotherapy-induced bowel injury. In some embodiments is provided a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of antibiotics-induced diarrhoea. In some embodiments is provided a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of antibiotics-induced radiation-induced bowel injury.

[0062] Also provided is a method of treatment or prevention of

[0063] a. chemotherapy-induced bowel injury;

[0064] b. antibiotics-induced diarrhoea; or

[0065] c. radiation-induced bowel injury,

[0066] said method comprising administering to a subject in need thereof an effective amount of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath).

[0067] Additionally provided is the use of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for the manufacture of a medicament for treatment of:

[0068] a. chemotherapy-induced bowel injury;

[0069] b. antibiotics-induced diarrhoea; orP7524EP00

[0070] c. radiation-induced bowel injury.

[0071] In some embodiments, said treatment is prophylactic. In some embodiments, said treatment is curative. In some embodiments, said treatment is ameliorating.

[0072] Said chemotherapy-induced bowel injury may be in the small and / or in the large intestine. Said radiation-induced bowel injury may also be in the small and / or in the large intestine.

[0073] The lysates of Methylococcus capsulates (Bath) according to the present disclosure may be prepared by any useful methods known in the art for preparation of bacterial lysates. Said lysate may e.g. be prepared as described in Jensen et al., 2021.

[0074] For example, in some embodiments, said lysate is prepared according to the following protocol: Methylococcus capsulatus (Bath) (McB) is cultivated in nitrate mineral salts (NMS) medium to produce a single-strain bacterial lysate. NMS medium is composed of 5 stock solutions; 10* NMS Salts (98.9 mM KNO3, 43.8 mM MgSO4*6H2O and 9.0 mM CaCI2), 1000x NaMoO4*2H20 (1.07 mM), 10,000xFeEDTA (103 mM), 1000x Trace Elements Solution (0.8 mM CuSO4*5H20,1.8 mM FeSO4*7H20, 1.4 mM ZnSO4*7H20, 0.24 mM H3BO3, 0.21 mMCoCI2*6H20, 0.74 mM EDTA-Na2, 0.1 mM MnCI2*4H20, 42.07 uM NiCI2*6H20)and 10x phosphate buffer pH 6.8 (198.25 mM Na2HPO4*12H20, 191.05 mMKH2PO4). When preparing 1x NMS medium from stock solutions 10x NMS Salts is added to 50% of the final volume with H2O before the remaining stock solutions are added. Then pH is adjusted to 6.8 before the NMS medium is sterile filtered using a 0.45-uM filter. Medium is stored in the dark. The 1000x Trace Element Solution is made from the following stock solutions and stored in the dark; 400.5 mM CuSO4*5H20, 3.6 mM FeSO4*7H20 (pH 3.0), 347.8 mM ZnSO4*7H20, 161.7 mM H3BO3, 42.0 mM CoCI2*6H20, 50.5 mM MnCI2*4H20 and 42.0 mM NiCI2*6H20. McB culture aliquots may be frozen in liquid nitrogen and stored at -80 °C. Cultivations on agar plates and in shake flasks (orbital shaker incubator at 200 rpm) are performed at 45 °C in an atmosphere of 75% air, 23.25% CH4, and 1.25% CO2. Continuous cultivation is carried out in a 3 L bioreactor (Applikon, The Netherlands) with a working volume of 2 L. Cells are precultivated in shake flasks and used to inoculate the bioreactor to an optical density at 440 nm (OD440) of 0.1. The temperature is maintained at 45 °C, stirring set to 650 rpm, and pH maintained at 6.8P7524EP00

[0075] by automatic addition of 2.5 M NaOH / 2.5 M HCI. A gas mixture of 75% air and 25% methane is sparged into the bioreactor. The continuous culture is started after an initial batch phase, and the dilution rate is set to 0.01 h-1. The OD440 at steady state is generally sustained at ~10. Culture effluent is collected, and cells are harvested by centrifugation. Bacterial cell walls are disrupted by the use of a French press before freeze-drying of the material.

[0076] Compositions

[0077] In some embodiments, the lysate in the composition as disclosed herein is a whole-cell lysate.

[0078] In some embodiments, the composition further comprises one or more active agents. In some embodiments, one or more of said active agents are prebiotics. In some embodiments, one or more of said active agents are probiotics. In some embodiments, said active agents comprise both prebiotics and probiotics.

[0079] In some embodiments, the composition comprises helper bacteria. “Helper bacteria” refer to bacterial species or strains that support or promote gut health, nutrient absorption, and / or the body's overall health through e.g. microbiome modulation.

[0080] Examples include bacteria of the genera Lactobacillus or Bifidobacterium. In some embodiments, the composition comprises from 1% to 100% helper bacteria, such as from 1% to 90%, such as from 1% to 80%, such as from 1% to 70%, such as from 1% to 60%, such as from 1% to 50%, such as from 1% to 40%, such as from 1% to 30%, such as from 1% to 20%, such as from 1% to 10% helper bacteria. In some embodiments, the composition comprises at the most 30%, such as at the most 20%, such as at the most 10%, such as at the most 5% helper bacteria.

[0081] Effects of administration of compositions

[0082] Administration of said composition to a subject suffering from chemotherapy-induced bowel injury, antibiotics-induced diarrhoea, or radiation-induced bowel injury may provide several beneficial effects to said subject, e.g. ameliorating or curing the symptoms associated with said conditions.

[0083] In some embodiments, administration of said composition to a subject results in decreased bowel frequency, decreased abdominal pain, reduced fecal calprotectinP7524EP00

[0084] levels, improved stool consistency according to the Bristol Stool Scale, reduced fecal and / or plasma lipocalin-2, and / or reduced fecal and / or plasma C-reactive protein. In some embodiments, administration of the composition as described herein to a subject results in decreased bowel frequency. In some embodiments, administration of the composition as described herein to a subject results in decreased abdominal pain. In some embodiments, administration of the composition as described herein to a subject results in reduced fecal calprotectin levels. In some embodiments, administration of the composition as described herein to a subject results in improved stool consistency according to the Bristol Stool Scale. In some embodiments, administration of the composition as described herein to a subject results in reduced fecal and / or plasma lipocalin-2 levels. In some embodiments, administration of the composition as described herein to a subject results in reduced fecal and / or plasma C-reactive protein levels.

[0085] Administration of the compositions as disclosed herein to a subject may increase the number of peripherally-induced regulatory T cells, which are useful for suppressing aberrant immune responses and may thus ameliorate, prevent, or cure etiologies or symptoms of gastrointestinal barrier dysfunction resulting from chemotherapy, antibiotics, or radiation treatment.

[0086] In some embodiments, administration of the composition as disclosed herein to a subject increases the number of peripherally-induced regulatory T cells by at least 1.5-fold, such as at least 2-fold, such as at least 2.5-fold, such as at least 3-fold, such as at least 3.5-fold, or such as at least 4-fold. In some embodiments, said peripherally-induced regulatory T cells are located in the small intestine. In specific embodiments, said peripherally-induced regulatory T cells are located in the lamina propria of the small intestine. In some embodiments, said increase in the number of peripherally-induced regulatory T is measured at the most 2 weeks after administration of said composition to said subject, such as at the most 10 days, such as at the most 1 week, such as at the most 5 days, such as at the most 4 days, such as at the most 3 days, such as at the most 2 days, such as at the most 1 day after administration of said composition to said subject.

[0087] Administration of the compositions as disclosed herein to a subject may increase the number of T-helper 17 (Th17) cells in the small and / or large intestine, particularly in theP7524EP00

[0088] small intestine. Without being bound by theory, said Th17 cells may have immunosuppressive and / or epithelial barrier-strengthening effects on the local microenvironment. Stimulation of these cells may therefore be useful for useful for suppressing aberrant immune responses and restoring a disrupted gut barrier and may thus ameliorate, prevent, or cure etiologies or symptoms of gastrointestinal barrier dysfunction resulting from chemotherapy, antibiotics, or radiation treatment.

[0089] In some embodiments, administration of the composition as disclosed herein to a subject increases the number of Th17 cells in the intestine, such as in the small intestine, by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 150%, such at least 200%, or such as at least 250%. In some embodiments, said increase in the number of Th17 cells is measured at the most 2 weeks after administration of said composition to said subject, such as at the most 10 days, such as at the most 1 week, such as at the most 5 days, such as at the most 4 days, such as at the most 3 days, such as at the most 2 days, such as at the most 1 day after administration of said composition to said subject.

[0090] Without being bound by theory, high levels of transforming growth factor p (TGF-p) and IL-27 can promote expression of anti-inflammatory cytokines by Th17 cells, including IL-10, alongside IL-17, whereas IL-1 p can promote transdifferentiation of Th17 cells to T-helper 1 (Thl)-like states, allowing for expression of pro-inflammatory interferon y (IFN-Y).

[0091] It is desirable when treating gastrointestinal barrier dysfunction resulting from chemotherapy, antibiotics, or radiation treatment that the Th17 cells induced by the compositions as disclosed herein have an immunosuppressive, barrier-strengthening profile, and e.g. express anti-inflammatory cytokines, such as IL-10, instead of having a pro-inflammatory profile, e.g. by expressing pro-inflammatory cytokines, such as IFN-y, or that the composition as disclosed herein cause a reduction the number of Th17 cells with a pro-inflammatory profile.P7524EP00

[0092] Thus, in some embodiments, administration of the composition as disclosed herein to a subject increases the number of I L-10+Th17 cells in the intestine, such as in the small intestine, by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 150%, such at least 200%, or such as at least 250%. In some embodiments, said increase in the number of I L-10+Th17 cells is measured at the most 2 weeks after administration of said composition to said subject, such as at the most 10 days, such as at the most 1 week, such as at the most 5 days, such as at the most 4 days, such as at the most 3 days, such as at the most 2 days, such as at the most 1 day after administration of said composition to said subject.

[0093] In some embodiments, administration of said composition to a subject decreases the number of IFN-y+Th17 cells in the intestine, such as in the small intestine, by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, or such as at least 100%. In some embodiments, said decrease in the number of IFN-y+Th17 cells is measured at the most 2 weeks after administration of said composition to said subject, such as at the most 10 days, such as at the most 1 week, such as at the most 5 days, such as at the most 4 days, such as at the most 3 days, such as at the most 2 days, such as at the most 1 day after administration of said composition to said subject.

[0094] In some embodiments, administration of said composition to a subject increases the ratio of IL-10+Th17 cells to IFN-y+Th17 cells in the intestine, such as in the small intestine. Said increase in ratio is preferably statistically significant. In some embodiments, said increase in the ratio is at least 1.5-fold relative to a baseline measurement. In some embodiments, said increase in the ratio is at least 2-fold relative to a baseline measurement. In some embodiments, said increase in the ratio of I L-10+Th17 cells to IFN-y+Th17 cells is measured at the most 2 weeks after administration of said composition to said subject, such as at the most 10 days, such as at the most 1 week, such as at the most 5 days, such as at the most 4 days, such asP7524EP00

[0095] at the most 3 days, such as at the most 2 days, such as at the most 1 day after administration of said composition to said subject.

[0096] Administration of said composition to a subject may thus restore, such as significantly restore, small and / or large intestinal barrier function. Said restoration of intestinal barrier function may be measured by any useful method known to the skilled person in the art, such as by oral lactulose or mannitol challenge test. In some embodiments, administration of said composition to a subject restores, such as significantly restores, small intestinal barrier function. In some embodiments, administration of said composition to a subject restores, such as significantly restores, large intestinal barrier function. In some embodiments, administration of said composition to a subject restores, such as significantly restores, small and large intestinal barrier function. In some embodiments, administration of said composition to a subject restores, such as significantly restores, intestinal barrier function in the duodenum, jejunum, and / or ileum. In some embodiments, administration of said composition to a subject restores, such as significantly restores, intestinal barrier function in the cecum, ascending colon, transverse colon, descending colon, sigmoid colon and / or rectum.

[0097] In some embodiments, administration of said composition to a subject reduces inflammation. In some embodiments, administration of said composition to a subject reduces inflammation in the small and / or large intestine. In some embodiments, administration of said composition to a subject reduces colitis associated with intestinal, such as colonic, barrier dysfunction.

[0098] Administration of compositions with other treatments

[0099] The compositions as disclosed herein may be administered together, i.e. concurrently, with the treatment that induces the gastrointestinal barrier dysfunction, e.g. in order to lessen or prevent the induction of said gastrointestinal barrier dysfunction. It may also be administered after, i.e. sequentially to, administration of the treatment that induces the gastrointestinal barrier dysfunction, e.g. in order to lessen the induction of or ameliorate said gastrointestinal barrier dysfunction. Said treatment may be chemotherapy- or radiation-based treatment of diseases, such as cancer, or administration of antibiotics, such as broad-spectrum antibiotics, for treatment of e.g. microbial infections, such as bacterial infections.P7524EP00

[0100] Thus, in some embodiments, the composition as disclosed herein is administered concurrently with or sequentially to a chemotherapeutic agent and / or a radiation treatment. In some embodiments, the composition as disclosed herein is administered concurrently with a chemotherapeutic agent and / or a radiation treatment. In some embodiments, the composition as disclosed herein is administered sequentially to a chemotherapeutic agent and / or a radiation treatment.

[0101] In some embodiments, the composition as disclosed herein is administered concurrently with or sequentially to one or more antibiotics, such as one or more broadspectrum antibiotics. In some embodiments, the composition as disclosed herein is administered concurrently with one or more antibiotics, such as one or more broadspectrum antibiotics. In some embodiments, the composition as disclosed herein is administered sequentially to one or more antibiotics, such as one or more broadspectrum antibiotics.

[0102] Formulation of compositions

[0103] The compositions as disclosed herein may be formulated in any useful manner known to the person skilled in the art in order to provide the lysate of the soil bacterium Methylococcus capsulates (Bath) to the site or environment at risk of, or already presenting with, gastrointestinal barrier dysfunction.

[0104] In some embodiments, the composition is formulated for oral administration. In some embodiments, the lysate is formulated in a tablet. In some embodiments, the lysate is formulated in a powder. In some embodiments, the lysate is formulated in a granule. In some embodiments, the lysate is formulated in a pellet. In some embodiments, the lysate is formulated in a capsule. In some embodiments, the lysate is formulated in a gel or paste. In some embodiments, the lysate is formulated or in a liquid, such as in a solution, dispersion, suspension or emulsion.

[0105] In some embodiments, the lysate is formulated as a food additive. In some embodiments, the lysate is formulated as a source of protein for use in a foodstuff, such as for use in an energy bar.P7524EP00

[0106] In some embodiments, the lysate is encapsulated. In some embodiments, the lysate is encapsulated in a microparticle, nanoparticle, or liposome.

[0107] In some embodiments, the lysate is sustainably released after administration. The skilled person knows how to formulate compounds to ensure stable, sustained and long-term release after administration, such as after oral administration. In some embodiments, the lysate is encapsuled in a sustained-release carrier.

[0108] Kits

[0109] In one aspect of the present disclosure is provided a kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating chemotherapy-induced bowel injury, antibiotic-induced diarrhea and / or radiation-induced bowel injury.

[0110] In some embodiments is provided a kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating chemotherapy-induced bowel injury.

[0111] In some embodiments is provided a kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating antibiotic-induced diarrhea.

[0112] In some embodiments is provided a kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating radiation-induced bowel injury.

[0113] In some embodiments, the composition is as described elsewhere herein.

[0114] Items

[0115] 1. A composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of:

[0116] a. chemotherapy-induced bowel injury;

[0117] b. antibiotics-induced diarrhoea; or

[0118] c. radiation-induced bowel injury.P7524EP00

[0119] 2. The composition for use according to any one of the preceding items, wherein said lysate is a whole-cell lysate.

[0120] 3. The composition for use according to any one of the preceding items, wherein said composition further comprises one or more active agents.

[0121] 4. The composition for use according to any one of the preceding items, wherein said composition further comprises one or more prebiotics and / or one or more probiotics.

[0122] 5. The composition for use according to any one of the preceding items, wherein said composition comprises from 1% to 100% helper bacteria, such as from 1% to 80%, such as from 1% to 60%, such as from 1% to 40%, such as from 1% to 20%, such as from 1% to 10% helper bacteria, such as at the most 10%, such as at the most 5% helper bacteria.

[0123] 6. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject results in decreased bowel frequency, decreased abdominal pain, reduced fecal calprotectin levels, improved stool consistency according to the Bristol Stool Scale, reduced fecal and / or plasma lipocalin-2, and / or reduced fecal and / or plasma C-reactive protein.

[0124] 7. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject increases the number of peripherally-induced regulatory T cells, such as increases the number of peripherally-induced regulatory T cells by at least 2-fold, such as at least 2.5- fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold.

[0125] 8. The composition for use according to item 7, wherein said T cells are located in the small intestine, such as in the lamina propria of the small intestine.

[0126] 9. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject increases the number of Th17P7524EP00

[0127] cells in the small intestine by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0128] 10. The composition for use according to item 9, wherein said Th17 cells are IL-10+Th17 cells.

[0129] 11. The composition for use according to any one the preceding items, wherein administration of said composition to a subject decreases the number of IFN-y+Th17 cells by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0130] 12. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject activates, such as increases activation of, glucagon-like peptide-2 receptors (GLP-2R) in the small and / or large intestine.

[0131] 13. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject restores small and / or large intestinal barrier function.

[0132] 14. The composition for use according to any one of the preceding items, wherein administration of said composition to a subject reduces inflammation, such as reduces colitis associated with colonic barrier dysfunction.

[0133] 15. The composition for use according to any one of the preceding items, wherein said chemotherapy-induced and / or radiation-induced bowel injury is in the small or large intestine.

[0134] 16. The composition for use according to any one of the preceding items, wherein said composition is administered concurrently or sequentially with a chemotherapeutic agent and / or a radiation treatment.

[0135] 17. The composition for use according to any one of the preceding items, wherein said composition is administered concurrently or sequentially with one or moreP7524EP00

[0136] antibiotics.

[0137] 18. The composition for use according to any of the preceding items, wherein said treatment is prophylactic, curative or ameliorating.

[0138] 19. The composition for use according to any one of the preceding items, wherein said composition is formulated for oral administration.

[0139] 20. The composition for use according to any one of the preceding items, wherein said lysate is formulated in a tablet, in a powder, in a granule, in a pellet, in a capsule, in a gel or paste, or in a liquid, such as in a solution, dispersion, suspension or emulsion.

[0140] 21. The composition for use according to any one of the preceding items, wherein said lysate is formulated as a food additive, such as a source of protein for use in a foodstuff, such as an energy bar.

[0141] 22. The composition for use according to any one of the preceding items, wherein said lysate is encapsulated, such as in a microparticle, nanoparticle, or liposome.

[0142] 23. The composition for use according to any one of the preceding items wherein said lysate is sustainably released after administration, such as wherein said lysate is encapsuled in a sustained-release carrier.

[0143] 24. A method of treatment or prevention of

[0144] a. chemotherapy-induced bowel injury;

[0145] b. antibiotics-induced diarrhoea; or

[0146] c. radiation-induced bowel injury,

[0147] said method comprising administering to a subject in need thereof an effective amount of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath).

[0148] 25. Use of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for the manufacture of a medicament for treatment of:P7524EP00

[0149] a. chemotherapy-induced bowel injury;

[0150] b. antibiotics-induced diarrhoea; or

[0151] c. radiation-induced bowel injury.

[0152] 26. A kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating chemotherapy-induced bowel injury, antibiotics-induced diarrhea, and / or radiation-induced bowel injury.

[0153] 27. The method according to item 24, the use according to item 25, or the kit according to item 26, wherein said composition is according to any one of items 1 to 23.P7524EP00

[0154] Examples

[0155] Materials and methods

[0156] Rodents and ethical statements

[0157] Animal experiments were approved by the Danish Animal Experiments Inspectorate (#2021-15-0201-01031 and # 2023-15-0201-01409). Six- to seven-week-old male and female C57BL / 6 mice and male Wistar rats were purchased from vendors as detailed below, and GLP-1R and GLP-2R KO mice were bred in-house. Upon arrival mice and rats were allowed to acclimatize in the animal facility environment for two weeks prior to study initiation. All rodents were housed under specific pathogen free conditions in 12h light / dark cycle (6AM-6PM). Male mice were housed 3 mice per cage and female mice were housed 5 per cage, unless otherwise specified below for each protocol. Rats were housed four per cage.

[0158] Diets and experimental setups

[0159] All special diets were obtained from Sniff Spezialdiaten GmBH, Germany, and stored at -20°C. Several customized diets were designed depending on the specific research question. In all protocols mice were fed ad libitum, and during termination mice were anesthetized with 2.5% isoflurane and euthanized by cervical dislocation following cardiac puncture blood collection. Cardiac blood samples were taken using EDTA coated needles and syringes. Plasma was collected by centrifugation of blood samples for 10 minutes at 1000 ref at 4°C and subsequently stored at -80°C.

[0160] Nutrient comparison protocol

[0161] Male mice were purchased from Taconic Laboratories, Denmark. After two weeks of acclimatization mice were given one of two diets, a diet with few simple sources of protein, fat, and carbohydrates (referred to as Simple Diet, SD) or an isocaloric macronutrient-matched diet with a complex composition of humanized sources of protein, fat, and carbohydrates (referred to as Complex Diet, CD) for eight weeks. At week eight a subgroup of mice was maintained on the respective simple or complex diets or fed an experimental diet with the protein source exchanged with solely McB lysates (contain <5% biomass from helper bacteria, sold under the commercial name Feedkind®, Calysta UK Ltd) (SDMCB or CDMCB) for an additional 4 weeks. To decouple any secondary effect of caloric density we also modified the complex low-fat- low-fiberP7524EP00

[0162] reference (referred to as Complexref) to also contain McB lysate as the main protein source.

[0163] Microbiota-dependency protocol

[0164] Male mice were purchased from Taconic Laboratories (Denmark). After two weeks of acclimatization, mice were split into two groups: one group given control drinking water and one group given a broad-spectrum antibiotics cocktail of 0.5 g / L Neomycin (Sigma #N1876-25G) and 1 g / L Ampicillin (Sigma #A9518-25G) in the drinking water to reduce gut microbial load. After acclimatization, mice underwent dietary intervention and were further split into two groups per drinking water condition: one group switched to the CD, and one group switched to the CDMCB. Mice were maintained on control drinking water or antibiotics during the dietary intervention period. Mice were then euthanized after two, four, or six weeks to look at the temporal effects of McB-consumption on blunted gut microbiota and mucosal T-cell populations.

[0165] Intestinal mucositis protocols

[0166] Female mice were purchased from Janvier (Le Genest-Saint-lsle, France), and female GLP-1R' / _and GLP-2R' / _mice were bred in-house. After at least a week of acclimatization, mice were fed either the Complexref or ComplexwcB for up to 13 days. After a week of dietary intervention, at experimental day 0, mice were intraperitoneally injected once with control saline or 5-fluorouracil (5-FU, Hospira Nordic AB, Stockholm, Sweden) at 400mg / kg to induce intestinal mucositis. Body weight development was subsequently monitored daily. Mice were euthanized at post-injection days 3, 4, or 6 (as indicated in figure legends), to investigate the acute and recovery phases of 5-FU-induced mucositis. Occasionally, mice were single-housed due to fighting and otherwise housed between 2-5 per cage. WT and KO mice were littermates.

[0167] Inhibition of microbial fermentation protocol

[0168] Male mice were ordered from Janvier (Le Genest-Saint-lsle, France) and allowed to acclimatize for at least a week. First, mice were fed the reference Complexref or ComplexMcB for one week and split into two cohorts: one subjected to colitis induction, and one for interrogation of lamina propria T-cells. Colitis was induced as described above. To inhibit microbial hindgut fermentation mice were given a 20 ppm solution of beta-acid extracts from Humulus lupulus as previously described in Singh, V. et al 2016, or control drinking water. For the colitis cohort, body weight and disease activity wereP7524EP00

[0169] monitored daily, and at day five mice were euthanized and tissues harvested. To test if lamina propria T-cells were dependent on microbial fermentation of McB, both numerically and phenotypically, T-cell subsets were studied using flow cytometry as described below.

[0170] Measurements of GLP-1 and GLP-2 and perfusion of rat intestine

[0171] 8-week-old Male Wistar rats were purchased from Janvier (Le Genest-Saint-lsle, France), Denmark, and allowed to acclimatize for 1 week. Rats were gavaged with a 5% w / v undigested McB lysate or a control protein source (casein, Sigma #03400) solution with PBS. Blood was collected by sublingual bleeding after 0, 7, 15, 30, and 45 minutes after gavage, to measure blood glycemia and circulating levels of GLP-1. After a washout period of 2 weeks, rat small intestines were perfused using a PBS-based 5% w / v lysate or control protein solutions as described previously (Modvig et al., 2021). Vascular effluents were collected every minute. GLP-1, and by proxy GLP-2, and amino acids were measured using radio-immuno-assay (RIA) as described previously (Modvig et al., 2021).

[0172] Measurements of GLP-2 in mice

[0173] C57BL / 6J were purchased from Janvier (Le Genest-Saint-lsle, France) and allowed to acclimatize for 1 week. Mice were gavaged with an inhibitor cocktail consisting of sacubitril (neprilysin inhibitor, 0.3 mg / kg, 5uL / g, cat. no. 333-B1070, Nordic Biosite, Sweden) and sitagliptin (dipeptidyl peptidase-4 inhibitor, 10 mg / kg, 5 uL / g, Xelevia) 30 minutes before oral delivery of microbial McB lysates of control protein. At time point 0, mice were gavaged with a 20% w / v (200 mg / mL in sterile saline) solution of McB lysate, a control protein solution (Albumin Fraction V, cat. no. 1.12018.0500, Sigma-Aldrich) or glucose. Blood was collected by cardiac puncture after 0-, 5-, 10-, and 20-minutes post gavage. GLP-2 levels were measured by RIA as above.

[0174] Histology

[0175] Intestinal sections were fixed in 10% paraformaldehyde and embedded in paraffin following standard procedures. Tissue slides were stained with H&E and samples were randomized and blinded before histological analyses. For small intestinal sections, villus height was measured by identifying three villi with visible extrusion zone, to ensure measurement of full-length villi, from each sample, and measuring from the tip until the crypt border. Crypt depth of small and large intestinal samples was assessed byP7524EP00

[0176] measuring the depth of three full-length crypts per slide, only when the entire crypt epithelium was visible from the lamina muscularis mucosa to the intestinal lumen. Villus height and crypt depth were analyzed using the Zeiss Zen Desk Software, from three separate areas within each section and reported as an average of measurements from all individuals.

[0177] Isolation of small and large intestine lamina propria (LP) cells

[0178] In protocols involving flow cytometry, after removal of 1 cm gut sections for histology, the small intestine was flushed with cold 1X HBSS (Gibco) containing 15 mM HEPES (Thermo Fischer), and large intestine flushed with cold 1X PBS and feces mechanically removed. Peyer’s Patches were carefully removed from small intestine. Both small intestine and large intestine were opened longitudinally and cut into 1 cm pieces in 1X HBSS and 15mM HEPES. Gut pieces were washed thrice in prewarmed HBSS washbuffer containing 1X HBSS, 15 mM HEPES, 2.5% heat-inactivated (HIA) FBS, 1 mM sodium pyruvate, 10.000 U / rnL Penicillin / Streptomycin, 50 mg / mL Gentamycin, and 2mM EDTA. After each wash, samples were incubated at 37°C for 10 min during first incubation and 15 min during second and third incubation. During incubation steps, large intestine samples, but not small intestine samples, were shaken on an orbital shaker at 450 rpm. After each incubation step small intestine samples were vigorously shaken by hand for 10 seconds. Media containing cell debris and epithelial cells were discarded by filtration through a 250 pm nylon mesh. The remaining tissue was digested for 20-25 min at 37°C under magnetic stirring at 450 rpm in R10 medium (RPMI 1640 with L-glutamine, 1 mM sodium pyruvate, 15 mM HEPES, 10.000 U / rnL Penicillin / Streptomycin, 50 mg / mL Gentamycin, 10% HIA FBS) containing 1 mg / mL Collagenase P (Roche) and 30 pg / mL DNAse I (Roche). After digestion, samples were mechanically agitated, filtered through a 100 pm cell strainer, and centrifugated at 500 ref for 7 minutes at 4°C and supernatant removed. The cell pellets were resuspended in 40% isotonic Percoll (GE Healthcare) and LP cells were purified by density centrifugation using 40 / 70 % Percoll gradient, centrifuged at 800 ref for 20 min at room temperature, acceleration 5 and brake 0. The lymphocyte interface was collected in fresh R10 medium followed by centrifugation for 7 min at 500 ref at 4°C. Subsequently, the supernatant was removed, and the cell pellet was resuspended in R10 medium and transferred to 96 well plates to be used for ex vivo stimulation of LP cells and ensuing flow cytometry.P7524EP00

[0179] Ex vivo stimulation of LP cells and staining

[0180] Small intestine-LP and large intestine-LP cells were restimulated ex vivo in R10 medium as follows. 250 ng / mL PMA (Sigma-Aldrich) in combination with 0.5 pg / mL lonomycin (Sigma-Aldrich) was added to restimulate samples and incubated at 37°C and 5% CO2 for four hours. After one hour 10 pg / mL brefeldin A was added to all samples, and continued incubation. After stimulation, plates were centrifuged for 5 min at 500 ref at 4°C and subsequently washed with PBS. After additional centrifugation for 5 min at 500 ref at 4°C, cells were resuspended in PBS containing Live / Dead stain (Zombie UV) and placed at 4°C in the dark for 20 min. Cells were washed with MACS buffer (containing PBS, HIA FBS, EDTA) and centrifuged for 5 min at 500 ref at 4°C. Cells were resuspended in MACS buffer and surface stained with primary antibodies (see Table 1 below) for 30 min at 4°C in the dark followed by washing and centrifugation. Overnight intracellular staining with primary antibodies (see Table 1 below) was performed using the FoxP3 / Transcription Factor Staining Buffer Set from eBioscience according to manufacturer’s instructions.

[0181] Flow cytometry

[0182] Flow cytometry was carried out by standard procedures and data acquired using a LSRFortessa X-20 (BD Bioscience). Data was analyzed using the FlowJo Software (Tree Star).

[0183] Antibodies

[0184] Antibodies used in the present examples are specified in Table 1, below.

[0185] Table 1. Antibodies used in the present examples.

[0186]

[0187] P7524EP00

[0188]

[0189] Microbiome analyses and bioinformatics processing

[0190] DNA was extracted with NucleoSpin Soil kit (Macherey-Nagel) following the manufacturer's protocol. DNA libraries were prepared using the MGIEasy DNA Library Prep Kit (MGI) following the manufacturer’s protocol. The MGIEasy DNA Adapters-96 (Plate) Kit (MGI) was used for adapter ligation. DNA cleanup was performed with the MGIEasy DNA Clean Beads (MGI). Circularization of libraries was carried out using the MGIEasy Circularization Module V2.0 (MGI). Sequencing was performed on the DNBSEQ-G400RS platform using the G400-PE150 sequencing kit (MGI), generating paired-end reads of 150 bp. Taxonomic profiling was performed with MetaPhlAn 3.0, while functional profiling of microbial pathways was conducted using HLIMAnN 3.0 (Beghini et al., 2021).

[0191] Fecal Bacterial DNA extraction

[0192] Bacterial DNA was extracted with centrifuge processing using the MN Stool 96 kit (Machery Nagel). Purified DNA was quantified using a Nanodrop 2000 (Thermo Scientific). All samples were processed within one week of extraction and stored at -20°C.P7524EP00

[0193] Quantification of fecal bacterial load

[0194] Bacterial 16S rRNA gene copies were quantified by qPCR on a Light Cycler 480 II (Roche) using V4 region-specific Primers 505F (5’-GTGYCAGCMGCCGCGGTAA-3’ -SEQ ID NO: 1) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’ - SEQ ID NO: 2). The thermal cycling conditions started with a DNA-denaturation step at 95° C for 5 minutes, followed by 50 cycles of i) denaturation at 95°C for 10 seconds, ii) annealing at 60°C for 20 seconds, and iii) extension at 72°C for 20 seconds. All assays were run in triplicates. Results from the Nanodrop were used for the DNA quantification and adjusted according to the fecal input and represented as DNA per mg of feces. A 2-fold standard curve was produced by serially diluting the DNA pool (starting dilution: 1:20) in sterile MilliQ Water. As a proxy for bacterial abundance, qPCR threshold cycle (Ct) values were converted to estimated bacterial genomes present in 1 mg of feces.

[0195] Tissue myeloperoxidase (MPO) levels

[0196] Approximately 1 cm of gut was homogenized in 50 mM HTAB in 50mM K-Phosphate buffer for 6 minutes with a steel bead. Tissues were snap frozen on dry ice, thawed in water and homogenized again; this cycle was done a total of 3 times. After centrifugation (16000g, 30 minutes at RT), supernatant was collected and diluted 10x for MPO measurement. Samples were mixed with substrate buffer (O-dianisidine dihydrochloride in 50mM K-Phosphate buffer with 0.0005% H2O2) on a 96-well plate, in duplicates, and measurements were taken every 30 seconds for 5 minutes at 450nm. MPO activity was calculated using all readings and corrected for total protein content of each sample.

[0197] Statistical analyses

[0198] Statistical analyses were performed using GraphPad Prism software (version 10). All data are presented as means ± standard error of the mean (SEM), with individual data points included to illustrate data distribution. For comparisons between two groups, unpaired t-tests were conducted to assess statistical significance. When comparing more than three groups with a single independent variable, a one-way analysis of variance (ANOVA) was utilized, followed by Tukey's post hoc test to identify specific group differences. In instances where more than three groups with two independent variables were analyzed, a two-way ANOVA was performed to evaluate the effects of both independent variables and their interaction. A p-value of <0.05 was considered statistically significant.P7524EP00

[0199] Example 1 - McB Induce Rapid and Persistent Gut Microbiota Alterations

[0200] To address if McB feeding was diet-dependent, we compared the simple compositionally defined diet (SD) previously used, to a humanized diet with a complex nutrient mix (CD), both with and without McB lysate as protein source (Figure 1A). Fecal shotgun sequencing revealed rapid changes in gut microbiota composition already a few days after McB-feeding and regardless of background diet (SD versus CD). McB-induced changes to the microbial community remained stable after their initial shift. The observed changes highlight robust and reproducible McB-induced microbiota modulation even with a commercial product with minimal amount of helper bacteria (FeedKind®, Calysta UK) used here. McB presence explained 27% percent variance compared to only 10% percent from all other nutrients combined, with several species consistently regulated by McB across diets (Figure 1C-D). Of note, McB was barely, and only transiently, detectable (<0,01 %) in fecal samples of a few mice consuming lysate-containing diets (Figure 1C). The observed microbiota changes were accompanied by major changes to the metabolic potential of the McB-associated microbiota (Figure 1D; >460 differentially regulated pathways) already a few days after McB-feeding and regardless of background diet. Noteworthy metabolic pathways enriched in feces of McB-fed mice were fermentation processes, including metabolic processes leading to production of short-chain fatty acids (SCFAs).

[0201] Example 2 - Nutritional Immune Imprinting Exhibit Subset Specific Microbiota Dependence

[0202] We examined the lamina propria (LP) T-cell landscape, in both small and large intestine, to study regionalized McB-, microbe-, and diet-dependent effects, by flow cytometry. Independent of background diet, McB-feeding increased the small and large intestinal LP pTreg populations (2.5 and 4 times, respectively), including the relative proportion of triple-positive IL-17+pTregs (Figure 1F-G & 1J-K). Notably, McB-feeding also induced a -50% increase in Th17 cells exclusively in the small intestine (Figure 1H-I & 1L-M). Although Th17 cells originally were conceived as proinflammatory culprits, recent literature points towards their pleiotropic nature being instrumental for maintaining Gl immune balance and metabolic homeostasis upon high-fat diet feeding. Indeed, the McB-modulated Th17 landscape may be more immunoregulatory as we noticed an increase in I L-10+Th17 cells paralleled by a reduction of IFN-y+Th17 cells, suggesting a shift in balance towards tolerogenic immunity, in both small and largeP7524EP00

[0203] intestinal LP (Figure 1H-I & 1L-M). McB-induced immune imprinting seemed targeted towards RORyt+T cell subsets, as neither Th1 nor thymic-derived Treg (nTreg) populations were affected by McB-feeding.

[0204] As Gl pTreg and Th17 cell plasticity may be modulated by the gut microbiota, we next investigated if McB-induced immune alterations were driven by the substantial shift in microbial community structures following McB-feeding (Figure 1) or if McB-induced immune imprinting developed independently of resident gut microbes. To test this, we administered either control drinking water or a broad-spectrum antibiotic cocktail (ABX) for 2, 4, or 6 weeks, blunting the gut microbiota (Figure 2A). Despite ABX-induced disruption of gut homeostasis, as evident from the ABX-induced increases in cecal size (Figure 2B), McB instantly enhanced pTreg abundance in the small intestine of both ABX-treated and non-treated mice (Figure 2C). This trait was, however, not fully recapitulated in LP of the large intestine, but pTreg induction was notably accelerated in ABX-treated mice (Figure 2D). We read the delayed pT reg-induction in colons of conventional mice as a direct consequence of their microbial ‘buffer’ (i.e. , niche occupation) limiting direct McB-host interactions. Summarized, these data suggest that the McB lysate can directly increase LP pTregs.

[0205] We next turned our attention to the Th 17 cells, also notably affected by McB feeding (Figure 2E-F). In sharp contrast to the above-mentioned pTreg inductions, we here found that both numeric and phenotypic McB-mediated Th 17 alterations were highly dependent on a functional gut microbiota. Thus, in the small intestine of conventional mice, McB feeding induced a rapid >50% increase in Th17 cell proportions (Figure 3E-J). This effect was dampened the first 4 weeks of ABX treatment. The McB-mediated Th17 induction after 6 weeks of ABX treatment mirrors the gradual increase in total microbial load. This rise in bacterial load suggests a selective bloom of ABX-resistant species, possibly interacting with McB to drive the observed expansion of Th17 cells, even under sustained ABX exposure. Further corroborating the Th17 cells-gut microbiota dependence, we observed that even in the absence of a numeric increase (large intestine, Figure 2H-I), McB-feeding still facilitated a phenotypic shift, exemplified by a >2-fold increase in I L-10+Th17 cells, exclusively in conventional mice (Figure 2E-J).P7524EP00

[0206] Together, these data demonstrate that while pTreg induction occurs independently of gut microbiota composition, Th17 cell induction and phenotypic manipulation relies on complex host-diet-microbe interactions, suggesting that the impact of the gut microbiota on McB-induced immune regulation is subset specific.

[0207] Example 3 - McB Promote Gastrointestinal Homeostasis and Reduce Inflammation During ABX treatment, we noticed McB fed mice experienced less diarrhea and were seemingly protected against ABX-induced increase in cecum weight (Figure 2B). Both diarrhea and swollen, fluid-filled cecums are common side effects of ABX treatment (Shaughnessy et al., 2022), presumably explained by disruptions in gut barrier function and mucus integrity, along with diminished microbial load and concurrent lack of fiber fermentation. Collectively, these data suggest that McB feeding preserve gut barrier function and concomitantly enhance tolerogenic immunity in both the small and large intestine.

[0208] If true, such traits would provide a significant leap forward in our conceptual understanding of nutritional immune imprinting and potentially pave the way for novel strategies to alleviate Gl inflammation. We thus scrutinized the therapeutic potential by employing a two-pronged approach investigating if McB feeding could also protect against experimental diseases characterized by region-specific gut inflammation, assessed in a model of chemotherapy-induced mucositis that, despite precipitating pan-GI mucositis, has a pronounced bias towards the small intestine, causing villus atrophy.

[0209] Gl mucositis was induced by administration of a single intraperitoneal (i.p) injection of 5-fluororuracil (5-Fll; 400 mg / kg) (Figure 3A). 5-fluororuracil is a cytotoxic chemotherapy medication used to treat cancer. Mice given 5-Fll exhibited marked weight loss for three days (marking an acute phase response), followed by rapid weight regain until body weights normalized after six days (recovery phase) (Figure 3B).

[0210] Compared to the mice fed the reference diet, McB-fed mice recovered slightly faster (Figure 3B-C). As 5-Fll is known to cause pronounced villus destruction (Billeschou et al., 2021), we examined the phenotype of the small intestine. 5-Fll administration greatly reduced small intestinal wet weights (Figure 3D) and caused major atrophy of intestinal villi (Figure 3E-G). Interestingly, McB feeding partially protected against 5-FU-mediated damage from proximal to distal small intestine (Figure 3E-G and Figure 5).P7524EP00

[0211] Previous reports have shown a compensatory response to 5-Fll administration during recovery, with hyperproliferation of the intestinal crypt epithelium to regenerate the atrophied absorptive surface (Billeschou et al., 2021; Hytting-Andreasen et al., 2018; Taminiau et al., 1980). We only observed this counterresponse in mice fed the refence diet - all McB-fed mice had consistently deeper crypts independent of 5-Fll administration, suggesting that McB feeding protects against 5-FU-induced Gl mucositis independently of proliferation. Although the effects of 5-Fll administration was most prominent in the small intestine, we still observed notable reductions in colon length of 5-Fll challenged mice (Figure 3H), pointing towards mucosal inflammation, a trait that was effectively prevented by McB (Figure 3H).

[0212] Collectively, these data demonstrate the ability of the bacterial lysate to not only modulate Gl immunity and microbiota composition, but also to mitigate inflammation in both the small and large intestine, showing notable benefits in subjects exposed to chemotherapy or antibiotics. Due to the many similarities between the Gl injuries caused by radiation treatment compared to chemotherapy, particularly that both treatments target rapidly dividing cells, the above positive effects of McB treatment are expected to also apply to radiation treatment induced Gl injury.

[0213] Example 4 - McB Selectively Activates the GLP-2R to Mediate Gastrointestinal Protection

[0214] Intrigued by the robust protection against small and large intestinal inflammation by the McB lysate, we speculated that the observed effects on the intestinal epithelium could be dependent on the intestinotrophic gut hormone GLP-2 and its receptor, the GLP-2R. To test this initial hypothesis, we orally gavaged rats and perfused their small intestines with solutions of McB or control protein (casein to match the protein source of reference diets). As a proxy for GLP-2 secretion we measured levels of its co-secreted counterpart, GLP-1, in circulation following oral gavage and in the vascular output of the perfused intestinal model. As we did not observe an increased secretion of GLP-1 (and thus by extension GLP-2) in either secretion models, we turned our attention towards the GLP-2R and hypothesized an interaction between the GLP-2R and lysate components. We employed both Gl inflammatory models to mice lacking the GLP-2R, to investigate any regions-specific GLP-2R dependence, given the bias of GLP-2 towards the small intestine (Drucker et al., 1997; Brubaker et al., 2004).P7524EP00

[0215] To evaluate the potential involvement of the GLP-2R, we conducted 5-Fll experiments in GLP-2R WT and KO mice (Figure 4A). Similar to findings from Figure 3, GLP-2R WTs fed reference diet lost weight (Figure 4B), had dramatically reduced small intestinal weights (Fig 4C) and villus atrophy across small intestinal segments (Figure 4D-E, 40). Still, McB fed mice were protected against intestinal damage (Figure 4C-G, 4N and Figure 6 A-B). Myeloperoxidase (MPO), an enzyme released by neutrophils during inflammation, serves as a key indicator of gut barrier damage and local inflammation. In our study, 5-Fll treatment significantly elevated intestinal MPO levels in reference diet-fed mice (Figure 4F-G). Remarkably, McB-feeding dramatically reduced these elevated MPO levels, suggesting a potent protective effect against 5-FU-induced gut inflammation and barrier disruption. Interestingly, ablation of the GLP-2R removed the therapeutic effects of McB (Figure 4H-N), indicating that the GLP-2R is indeed involved in protection of the small intestine. As previously shown, 5-Fll not only damaged the small intestine, but also cause colonic alterations. Importantly, GLP-2R KO ameliorated the McB-mediated rescue of the 5-FU-induced colon damage (Figure S4), indicating that lysate-mediated protection against Gl inflammation via the GLP-2R is region independent.

[0216] Considering the co-secretion of GLP-2 and GLP-1, we speculated if pan-GI protection was solely dependent on the GLP-2R, or if the sibling receptor, the GLP-1 R, was partly involved in the McB-mediated protection. Strikingly, both GLP-1 R WT and GLP-1 R KO littermates, were fully protected against 5-FU-induced mucositis, corroborating selectivity for GLP-2R signaling in the elicited effects.

[0217] Together these data demonstrate that the McB-based alternative protein source interact with the host GLP-2R to protect against inflammation of the small and large intestine, preventing excessive villus atrophy during mucositis, which is seemingly independent of the mucosal pTregs and Th17 cells.

[0218] Example 5 - McB does not stimulate secretion of GLP-1 or GLP-2

[0219] Considering the dependency of the GLP-2R to confer gut-protection, we investigated whether oral delivery or direct intestinal perfusion stimulated secretion of gut hormones GLP-1 and GLP-2. We perfused the rat small intestines with solutions of McB or control protein (casein to match the protein source of reference diets) (Figure 7A). As a proxy for GLP-2 secretion we measured levels of its co-secreted counterpart, GLP-1, inP7524EP00

[0220] circulation following oral gavage and in the vascular output of the perfused intestinal model. As we did not observe an increased secretion of GLP-1 (and thus by extension GLP-2) (Figure 7B) or change in amino acid absorption (Figure 70), we decided to test if the delivery would be dependent on gastrointestinal processing. Since this model bypasses digestion, enzymatic processing could still be needed to liberate bioactive components stimulating GLP-2 secretion.

[0221] In order to investigate this, we gavaged mice with McB or a control protein mix following administration of inhibitors, and then measured plasma GLP-2 via radio-immuno-assay (RIA) (Figure 7D). While glucose stimulated GLP-2 release, neither McB nor the reference protein increased circulating GLP-2 (Figure 7E). These findings demonstrate that McB does not acutely induce endogenous GLP-2 secretion.

[0222] Example 6 - Microbial fermentation is needed for the McB-mediated gut protection Given the known effects of microbial fermentation products on gastrointestinal health, and our observation of enrichment of short-chain fatty acid (SCFA)-productive capacity, we speculated if microbial fermentation of McB could generate metabolites capable of either stimulating GLP-2 release or directly activating the GLP-2R via molecular mimicry as a prerequisite for its beneficial effects.

[0223] To test this hypothesis, we employed the DSS colitis model and administered a fermentation inhibitor (Fermlnh; 20 ppm beta-acid extract from Humulus I u pul us) to block microbial hindgut fermentation (Figure 8A). In reference-fed mice, Fermlnh had no significant effect on disease severity (Figure 8B-G). Consistent with prior findings, McB-fed mice exhibited reduced DAI scores, attenuated tissue inflammation, conserved colon length, and reduced histopathological scores compared to controls (Figure 8H-M). Strikingly, Fermlnh treatment generally abolished McB’s protective effects, exacerbating DAI scores (Figure 8H-I), elevating the tissue inflammation index (Figure 8J), and leading to intestinal shortening (Figure 8K) reflected by the aggravation of tissue pathology (Figure 8M). Given McB’s intestinotrophic effects in healthy mice, we assessed intestinal morphology. Fermlnh eliminated McB-induced elongation of both small and large intestines, even in healthy mice (Figure 8L-M), directly linking microbial fermentation to McB-mediated gut homeostasis.P7524EP00

[0224] Because fermentation yields microbial metabolites known to stimulate gut hormone secretion, we asked whether blocking fermentation would lower systemic GLP-2 levels. Despite extended McB feeding (~2 weeks) and thus enzymtic processing, we found no significant increase in circulating GLP-2 in either control or McB-fed mice, regardless of fermentation status (Figure 8N). Interestingly, the repeatedly demonstrated effect of McB on pTreg induction was not altered when microbial fermentation was inhibited (Figure 9).

[0225] Sequence overview

[0226]

[0227] References

[0228] Beghini, F. etal. Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. Elife 10, e65088 (2021) Billeschou, A. etal. Intestinal Adaptation upon Chemotherapy-Induced Intestinal Injury in Mice Depends on GLP-2 Receptor Activation. Biomedicines (2021) doi: 10.3390 / biomedicines9010046.

[0229] Brubaker, P. L. & Drucker, D. J. Minireview: Glucagon-Like Peptides Regulate Cell Proliferation and Apoptosis in the Pancreas, Gut, and Central Nervous System. Endocrinology 145, 2653-2659 (2004).

[0230] Chassaing, B. et al. Fecal Lipocalin 2, a Sensitive and Broadly Dynamic Non-lnvasive Biomarker for Intestinal Inflammation. PLoS One 7, e44328 (2012).

[0231] Drucker, D. J., Deforest, L. & Brubaker, P. L. Intestinal response to growth factors administered alone or in combination with human [Gly2]glucagon-like peptide 2. Am. J. Physiol. Liver Physiol. 273, G1252-G1262 (1997).

[0232] Hytting-Andreasen, R. etal. Endogenous glucagon-like peptide- 1 and 2 are essential for regeneration after acute intestinal injury in mice. PLoS One (2018) doi: 10.1371 / journal. pone.0198046.P7524EP00

[0233] Jensen, B.A.H., Holm, J.B., Larsen, I.S. etal. Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORyt+IL-17+Tregs and improve metabolism. Nat Commun 12, 1093 (2021).

[0234] https : / / do i . org / 10.1038 / s41467-021 -21408-9

[0235] Katakura, K. etal. Toll-like receptor 9-induced type I IFN protects mice from experimental colitis. J. Clin. Invest. 115, 695-702 (2005).

[0236] Kleiveland CR, Hult LT, Spetalen S, et al. The noncommensal bacterium Methylococcus capsulatus (Bath) ameliorates dextran sulfate (Sodium Salt)-lnduced Ulcerative Colitis by influencing mechanisms essential for maintenance of the colonic barrier function. Appl Environ Microbiol. 2013;79(1):48-56. doi:10.1128 / AEM.02464-12

[0237] Modvig, I. M. et al. Amino acids differ in their capacity to stimulate GLP-1 release from the perfused rat small intestine and stimulate secretion by different sensing mechanisms. Am. J.

[0238] Shaughnessy, M. P., Park, C. J., Salvi, P. S. & Cowles, R. A. Jejunoileal mucosal growth in mice with a limited microbiome. PLoS One 17, e0266251 (2022).

[0239] Taminiau, J. A., Gall, D. G. & Hamilton, J. R. Response of the rat smallintestine epithelium to methotrexate. Gut 21, 486 LP-492 (1980).

[0240] Singh, V. etal. Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer. Cell 175, 679-694. e22 (2018).

Claims

37P7524EP00Claims1. A composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for use in the treatment of:a. chemotherapy-induced bowel injury;b. antibiotics-induced diarrhoea; orc. radiation-induced bowel injury.

2. The composition for use according to any one of the preceding claims, wherein said lysate is a whole-cell lysate.

3. The composition for use according to any one of the preceding claims, wherein said composition further comprises one or more active agents, such as one or more prebiotics and / or one or more probiotics.

4. The composition for use according to any one of the preceding claims, wherein administration of said composition to a subject increases the number of peripherally-induced regulatory T cells, such as increases the number of peripherally-induced regulatory T cells by at least 2-fold, such as at least 2.5- fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold.

5. The composition for use according to claim 4, wherein said T cells are located in the small intestine, such as in the lamina propria of the small intestine.

6. The composition for use according to any one of the preceding claims, wherein administration of said composition to a subject increases the number of Th17 cells, such as IL-10+Th17 cells, in the small intestine by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

7. The composition for use according to any one the preceding claims, wherein administration of said composition to a subject decreases the number of IFN-y+Th17 cells by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.38P7524EP008. The composition for use according to any one of the preceding claims, wherein administration of said composition to a subject restores small and / or large intestinal barrier function.

9. The composition for use according to any one of the preceding claims, wherein administration of said composition to a subject reduces inflammation, such as reduces colitis associated with colonic barrier dysfunction.

10. The composition for use according to any one of the preceding claims, wherein said composition is administered concurrently or sequentially with a chemotherapeutic agent and / or a radiation treatmentand / orwherein said composition is administered concurrently or sequentially with one or more antibiotics.

11. The composition for use according to any of the preceding claims, wherein said treatment is prophylactic, curative or ameliorating.

12. The composition for use according to any one of the preceding claims, wherein said composition is formulated for oral administration.

13. The composition for use according to any one of the preceding claims, wherein said lysate is formulated as a food additive, such as a source of protein for use in a foodstuff, such as an energy bar.

14. Use of a composition comprising a lysate of the soil bacterium Methylococcus capsulates (Bath) for the manufacture of a medicament for treatment of:a. chemotherapy-induced bowel injury;b. antibiotics-induced diarrhoea; orc. radiation-induced bowel injury,optionally wherein said composition is as defined in any one of claims 1 to 13.

15. A kit comprising a composition comprising a lysate of Methylococcus capsulatus (Bath) and instructions for use for treating chemotherapy-inducedP7524EP00bowel injury, antibiotics-induced diarrhea, and / or radiation-induced bowel injury, optionally wherein said composition is as defined in any one of claims 1 to 13.

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