Akkermansia for the prevention and / or treatment of reward system dysregulation

Abstract

The present invention relates to a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof for use in the prevention and / or treatment of reward system dysregulation.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of disorders related to reward system dysregulation.In particular, the present invention relates to a composition comprising one or more bacteria of the genus Akkermansia and / or its extract and / or fragment for use in preventing and / or treating reward system dysregulation. [Background technology]

[0002] Obesity, whose prevalence has steadily increased in recent decades, is strongly associated with excessive food intake, biased towards energy-dense foods. This food intake, based on hedonic value, is controlled by the food reward system. Palatable foods rich in fat and / or sugar stimulate the hedonic, reinforcing and motivational processes of the reward system (Berland C et al, Cell Metab, 2020).

[0003] Dopaminergic neurons in the mesocorticolimbic region of the brain are stimulated by palatable food and release dopamine from the ventral tegmental area (VTA) to the striatum (Str), including the nucleus accumbens (Nacc). Chronic overeating is associated with decreased dopamine release, decreased expression of dopamine receptor proteins (DRD2 and DRD1) and increased expression of dopamine transporter protein (DAT) (Volkow ND et al, Philos Trans R Soc Lond B Biol Sci, 2008). This reduced function of the dopamine pathway, in turn, leads to alterations in hedonic and motivated eating behavior (Wang GJ et al, Lancet, 2001).

[0004] Additionally, food intake is mediated through the brain reward system, which, when dysregulated, leads to eating disorders (Avena and Bocarsly, Neuropharmacology, 2012; Frank, Curr Psychiatry Rep, 2013). Similarly, drug reward has been shown to overlap with food reward circuitry, which, when dysregulated, can lead to addiction (Volkow, et al., Curr Topics Behav Neurosci, 2012; Rogers, Pharmacology, Biochemistry and Behavior, 2017).

[0005] The gut microbiota is a key regulator of host metabolism, including hypothalamic regulation of food intake via the gut-brain axis (Cani PD et al, Nat Metab, 2019; van de Wouw M et al, J Nutr, 2017). In obesity, gut microbiota composition is altered and intestinal permeability is increased. This allows bacterial components such as LPS (lipopolysaccharide) to cross the intestinal barrier and enter the systemic circulation, a condition called metabolic endotoxemia (Cani PD et al, Diabetes, 2007). LPS can induce inflammation in several organs, including the brain, by inducing the c-Jun N-terminal kinase (JNK) inflammatory pathway through nuclear transcription factor kappa B (NFκB) and TLR4, which is associated with the disruption of the blood-brain barrier (Zhao J et al, Sci Rep, 2019).

[0006] A causal role of the gut microbiota in the dysregulated reward system in the context of obesity, and more specifically, in the reward and hedonic components of food intake, has recently been demonstrated (de Wouters d'Oplinter A et al,Gut Microbes,2021). One consistent gut microbiota imbalance associated with obesity is a reduced abundance of Akkermansia muciniphila (A. muciniphila). Supplementation with A. muciniphila allows the prevention of weight gain and metabolic disorders in rodents and humans (Plovier H et al,Nat Med,2017; Depommier C et al,Nat Med,2019).

[0007] To date, the treatment of reward system dysregulation has been primarily provided by neuropharmacological compounds and / or psychotherapy. Therefore, there is a need to provide state-of-the-art, including alternative therapies for treating reward system dysregulation.

[0008] The present invention relates to the treatment and / or prevention of reward system dysregulation by utilizing the beneficial effects of A. muciniphila on neural and behavioral changes in the reward system. Summary of the Invention

[0009] The present invention relates to a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof for use in the prevention and / or treatment of reward system dysregulation.

[0010] In some embodiments, the bacterium is Akkermansia muciniphila or Akkermansia spp., and combinations thereof.

[0011] In some embodiments, the reward system dysregulation is selected from the group including or consisting of a psychiatric disorder, a neurological disorder, a disorder due to side effects of treatment, and combinations thereof.

[0012] In some embodiments, the psychiatric disorder is selected from the group including or consisting of an addiction-related disorder, an eating-related disorder, an emotional disorder, an obsessive-compulsive disorder, schizophrenia, attention-deficit hyperactivity disorder (ADHD), an autism spectrum disorder, an anxiety disorder, and the like.

[0013] In some embodiments, the addiction-related disorder is selected from the group including or consisting of alcohol-related addiction, drug-related addiction, gaming-related addiction, and the like.

[0014] In some embodiments, the neurological disorder is selected from the group including or consisting of Parkinson's disease, Tourette's syndrome, and the like.

[0015] In some embodiments, the disorder due to a side effect of treatment is selected from the group including or consisting of gaming addiction, shopping addiction, food addiction such as binge eating, hypersexuality, and the like.

[0016] In some embodiments, the eating disorder includes or is selected from the group consisting of bulimia nervosa, binge eating disorder, anorexia nervosa (including restrictive and binge eating / purging types), pica, rumination disorder, purging disorder, night eating syndrome, avoidant-restrictive food intake disorder, overweight-related disorders and obesity-related disorders, food addiction, eating addiction, food cravings, food seeking, compulsive eating disorder, impulsive eating disorder, failure of a calorie restricted diet, weight loss non-responder or non-responder to dietary advice for weight loss, and the like.

[0017] In some embodiments, the composition further comprises one or more active agents.

[0018] In some embodiments, the active agent is a therapeutic or nutritional agent.

[0019] In some embodiments, the active agent is a beneficial microorganism selected from the group including or consisting of bacteria from the family Verrucomicrobia, Tannerellaceae, Clostridiaceae, Peptostreptococcaceae, Prevotellaceae, Methylobacteriaceae, Parabacteroides, Turicibacter, Coprococcus, Knoellia, Prevotella, and Staphylococcus.

[0020] In some embodiments, the composition is in the form of a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0021] In some embodiments, the composition is in the form of a nutritional composition that further comprises a nutritionally acceptable carrier.

[0022] The present invention also relates to a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof for use as an adjuvant for a therapeutic agent administered to a subject suffering from a reward system dysregulation disorder.

[0023] In some embodiments, the composition is included in a kit, the kit further comprising a means for administering said composition.

[0024] definition For the purposes of the present invention, the following terms have the following meanings:

[0025] The term "about" preceding a value includes ±10% of the numerical value or less. It is to be understood that the value to which the term "about" refers is specifically, and preferably as such, disclosed.

[0026] "Adjuvant" refers to a component that enhances and / or modulates the immune response to an antigen toward a desired immune response. Benefits of adjuvants include enhancing the immunogenicity of antigens, improving the nature of the immune response, reducing the amount of antigen required for successful immunization, and improving the immune response in elderly and immunocompromised individuals.

[0027] "Bacteria of the genus Akkermansia" refers to Verrucomicrobia, Gram-negative anaerobic, non-spore-forming, non-motile bacteria. Bacteria belonging to the genus Akkermansia can be easily identified by routine procedures, including physiological and biochemical techniques, their cellular fatty acid profiles, menaquinone profiles, and assessment of their phylogenetic position based on whole-genome-based 16S rRNA gene sequence analysis, any tool that reveals subspecies-level genetic stratification, or inferential analysis using CRISPR-Cas loci. Examples of predicted Akkermansia species identified by such techniques are disclosed in Karcher N et al, Genome Biol, 2021.

[0028] "Beneficial microorganisms" refer to microorganisms that may provide health benefits to the host, including improving the host's gut microbial balance, maintaining intestinal barrier homeostasis, preventing pathogen colonization, and preventing bacterial and viral infections.

[0029] "Comprise" is intended to mean "contain," "encompass," and "include." In some embodiments, the term "comprise" also encompasses the term "consist of."

[0030] "Eating-related disorder" refers to a particular form of mental disorder in which the dysregulation of the reward system targets food intake, particularly the intake of palatable foods. Non-limiting examples of eating-related disorders include anorexia nervosa, bulimia nervosa, binge eating disorder, pica, rumination disorder, purging disorder, night eating syndrome, avoidant-restrictive food intake disorder, and overweight-related disorders. Eating-related disorders are often associated with abnormal body mass index (BMI), e.g., obesity. In one embodiment, anorexia nervosa includes restrictive type and binge eating / purging type.

[0031] An "enriched composition" refers to a composition in which the population density of bacteria of the genus Akkermansia is enhanced within the total microbial population of the composition.

[0032] "Extract" refers to any fraction obtained from the bacterium of interest or from the medium in which the bacterium of interest is cultivated. In practice, the extract includes cellular extracts and extracellular extracts. In one embodiment, the extract according to the invention includes metabolites from the bacterium.

[0033] As used herein, "fragment" refers to any part of the cell of the bacterium of the present invention. Preferably, said fragment is a membrane fraction obtained by membrane preparation. Membrane preparations of microorganisms belonging to the genus Akkermansia can be obtained by methods known in the art. Alternatively, whole cell preparations are also envisaged. Preferably, the fragments described herein of the microorganism of the present invention retain the ability to prevent and / or treat reward system dysregulation.

[0034] An "individual" or "subject" refers to an animal individual, preferably a mammalian individual, more preferably a human individual. In some embodiments, the individual may be a mammalian individual. Mammals include, but are not limited to, all primates (human and non-human), cows (including cows), horses, pigs, sheep, goats, dogs, cats, and any other mammals that are waiting to receive or are receiving medical care, or have been / are / will be the subject of a medical procedure, or are being monitored for the development of a reward system dysregulation. In some embodiments, the individual may be a "patient", i.e., a warm-blooded animal, more preferably a human, that is waiting to receive or are receiving medical care, or have been / are / will be the subject of a medical procedure, or are being monitored for the development of a reward system dysregulation. In some embodiments, the individual is an adult (e.g., an individual over 18 years of age). In some embodiments, the individual is a child (e.g., an individual under 18 years of age). In some embodiments, the individual is a male. In some embodiments, the individual is a female.

[0035] "Isolated bacteria" refers to bacteria that are no longer in their natural and / or physiological habitat or environment. For example, the bacteria of interest from the microbiota can be collected, separated from other bacteria, and further formulated in a composition. Bacterial isolation can be performed according to standard protocols in the field of microbiology, such as gram coloring, antibiotic resistance, ability to grow on specific substrates / media, and protocols adapted thereto.

[0036] "Mental disorders" refers to disorders characterized by a combination of abnormal thoughts, perceptions, emotions, behaviors, and relationships, as defined by the World Health Organization (WHO). In practice, mental disorders include addiction-related disorders, eating-related disorders, affective disorders, obsessive-compulsive disorders, schizophrenia, attention-deficit hyperactivity disorder (ADHD), autism spectrum disorders, anxiety disorders, etc.

[0037] "Neurological disorders" refer to disorders that affect the brain, nerves, and spinal cord. In practice, individuals with neuropathy may experience symptoms such as motor paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain, and altered levels of consciousness.

[0038] A "pharmaceutically acceptable carrier" refers to a carrier that does not produce any adverse, allergic or other undesirable reactions when administered to an animal individual, preferably a human individual. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. For human administration, preparations must meet sterility, pyrogenicity, general safety and purity standards as required by regulatory authorities, such as the United States Food and Drug Administration (FDA) or the European Union's European Medicines Agency (EMA).

[0039] "Prevention" refers to preventing or avoiding the occurrence of a symptom of reward system dysregulation. In the present invention, the term "prevention" may refer to secondary prevention, i.e., preventing the reoccurrence or relapse of a symptom of reward system dysregulation.

[0040] "Reward system dysregulation" refers to a disorder in which an individual's reward system does not generate a normal, sufficient or adapted response to rewarding stimuli, thereby resulting in an alteration in motivation or pleasure induction by the stimuli. Within the scope of this disclosure, an individual with reward system dysregulation has increased or decreased motivation and / or increased or decreased pleasure by rewarding stimuli. Typically, reward system dysregulation produces behavioral changes in the individual, promoting compulsive behavior. In practice, reward system dysregulation encompasses psychiatric and neurological disorders, which are defined below. Diagnosis of an individual with reward system dysregulation can be performed by a certified operator, such as a physician, according to standard protocols in the field, in particular by monitoring clinical signs, and often with the aid of a questionnaire.

[0041] "Therapeutically effective amount" refers to an amount sufficient to effect beneficial or desired results, including clinical outcomes. A therapeutically effective amount may be administered in one or more administrations. In one embodiment, the therapeutically effective amount may depend on the individual being treated.

[0042] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and preventative or prophylactic measures, the purpose of which is to prevent or slow (attenuate) the targeted reward system dysregulation. Those in need of treatment include subjects who already have a reward system dysregulation, as well as those who are prone to have a reward system dysregulation or those who should be prevented from having a reward system dysregulation. An individual or mammal is successfully "treated" for a reward system dysregulation or condition if, after administration of a therapeutic amount of a composition, pharmaceutical composition according to the present invention, alone or in combination with another therapeutic agent, the patient shows an observable and / or measurable reduction or absence of one or more symptoms associated with a reward system dysregulation and / or some relief of one or more symptoms associated with a reward system dysregulation or condition; reduced morbidity and mortality, and improved quality of life issues. The above parameters for assessing the success of treatment and improvement of the disease are easily measurable by routine procedures familiar to physicians.

[0043] Other definitions may appear within the context of the entire disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention relates to a composition comprising one or more bacteria of the genus Akkermansia and / or extracts or fragments thereof for use in the prevention and / or treatment of reward system dysregulation.

[0045] In some embodiments, the present invention also relates to the use of a composition comprising one or more bacteria of the genus Akkermansia and / or an extract or fragment thereof for preventing and / or treating reward system dysregulation.

[0046] The present invention further relates to the use of a composition comprising one or more bacteria of the genus Akkermansia and / or an extract or fragment thereof for preparing or manufacturing a medicament for the prevention and / or treatment of reward system regulation disorders.

[0047] In another aspect, the present invention relates to a method for preventing and / or treating reward system dysregulation, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising one or more bacteria of the genus Akkermansia and / or an extract or fragment thereof.

[0048] According to some embodiments, the Akkermansia bacterium is selected from the group including or consisting of Akkermansia muciniphila, Akkermansia glycaniphila, Akkermansia biwaensis, Akkermansia spp., and combinations thereof.

[0049] In some embodiments, the Akkermansia bacterium is Akkermansia muciniphila.

[0050] In fact, bacteria belonging to the genus Akkermansia can be identified by any suitable procedure or procedure adapted to them.In particular, suitable procedures can include physiological and biochemical methods, such as evaluation of fermentation ability with selected nutrients, such as mannose, raffinose; evaluation of resistance to some antibiotics; evaluation of specific enzyme activity, such as alpha-galactosidase, beta-galactosidase, alpha-glucuronidase, alkaline phosphatase, L-arginine, leucine glycine arylamidase, phenylalanine arylamidase; evaluation of their cellular fatty acid profile, menaquinone profile; evaluation of their profile by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); evaluation of their phylogenetic position based on genome-based 16S rRNA gene sequence analysis, any tool that reveals subspecies level genetic stratification, or inferential analysis using CRISPR-Cas loci.

[0051] In some embodiments, Akkermansia bacteria is isolated.In some embodiments, Akkermansia bacteria is isolated from natural environment, such as gut microbiota.In practice, Akkermansia bacteria can be isolated from feces or caecal contents, diluted or undiluted, fresh or frozen in specific medium (containing cryoprotectant and / or antioxidant), according to the standard and ethical procedures in this field.

[0052] In practice, bacteria of the genus Akkermansia may be cultured in any suitable medium, such as, for example, fastidious anaerobe broth (DSMZ®, commercially available from Neogen®), Pyg medium (modified) (commercially available from DSMZ®), Columbia broth (CB), brain heart infusion (BHI)-agar, etc., supplemented with mucus and optionally containing N-acetylglucosamine and / or N-acetylgalactosamine.

[0053] In practice, cultivation of Akkermansia bacteria may be carried out at a temperature of about 30° C. to about 42° C., preferably about 35° C. to about 40° C., and more preferably about 37° C. As used herein, the term "about 30° C. to about 42° C." includes about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., and 42° C.

[0054] In practice, the cultivation of bacteria of the genus Akkermansia is carried out under anaerobic conditions, ie in the absence of O2 or under partial oxygen depletion.

[0055] In some embodiments, a composition of the invention comprises, or consists essentially of, a microbiome having Akkermansia bacteria obtained from an individual.

[0056] In some embodiments, the compositions of the present invention are enriched for bacteria of the genus Akkermansia. In some embodiments, the compositions of the present invention comprise or essentially consist of a microbiota enriched for bacteria of the genus Akkermansia. In one embodiment, the microbiota is a gut microbiota obtained from the feces of an individual. In one embodiment, the microbiota is enriched for bacteria of the genus Akkermansia compared to the microbiota of the individual to be treated.

[0057] In practice, Akkermansia bacteria can be enriched by selectively stimulating the growth of Akkermansia bacteria.For example, enrichment can be performed by changing the physiological conditions of culture.Examples include, but are not limited to, changing the composition of medium, such as nutrient composition; and changing culture conditions, such as environmental pH value, temperature and oxygen conditions.

[0058] In some embodiments, the bacteria of the genus Akkermansia are isolated and enriched. In some embodiments, the compositions of the invention comprise isolated, enriched bacteria of the genus Akkermansia.

[0059] In some embodiments, the compositions of the invention comprise purified bacteria of the genus Akkermansia. The terms "purified" or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolate" refers to the degree of separation from its original source or surroundings. "Purify" refers to a degree of separation greater than isolation. Thus, purified isolated bacteria are at least about 90%, about 95%, about 99% or 100% free from other bacteria, fungi, viruses, or other unknown microorganisms.

[0060] In one embodiment, the Akkermansia bacteria is viable. As used herein, the term "viable" refers to bacteria that can maintain an active metabolism and / or grow in a suitable medium under suitable culture conditions, including suitable pH, temperature, salinity, nutrient content, and O2 content.

[0061] In one embodiment, the Akkermansia bacterium is non-viable, and said non-viable bacterium retains the ability to prevent and / or treat reward system dysregulation.As used herein, the term "non-viable" refers to a bacterium that cannot maintain active metabolism and / or cannot grow in a suitable medium under suitable culture conditions, including suitable pH, temperature, salt, nutrient content, and O2 content.Examples of non-viable bacteria are dormant bacteria, dead bacteria, and inactive bacteria.

[0062] In one embodiment, the Akkermansia bacteria is live. In another embodiment, the Akkermansia bacteria is dead, and said dead bacteria retains the ability to prevent and / or treat reward system dysregulation.

[0063] In practice, cell viability (active metabolism) can be evaluated by measuring the consumption of one nutrient in the medium over time.Cell viability (growth) can be evaluated by spreading a solution containing at least one of the bacteria of the present invention over a petri dish and counting the number of colonies after a given time of incubation in suitable culture conditions; alternatively, bacteria can be grown in a liquid medium and growth can be measured by measuring the optical density of the bacterial culture after a specified time of incubation in suitable culture conditions.

[0064] In one embodiment, the Akkermansia bacteria is pasteurized, and said pasteurized bacteria retain the ability to prevent and / or treat reward system dysregulation. In one embodiment, the pasteurized Akkermansia bacteria and / or extract thereof is heated at a temperature in the range of about 50°C to 100°C, preferably about 60°C to about 95°C, more preferably about 70°C to about 90°C, and even more preferably at a temperature of about 70°C.

[0065] In some embodiments, the Akkermansia bacteria is heat-inactivated and the heat-inactivated bacteria retain the ability to prevent and / or treat reward system dysregulation. In some other embodiments, the Akkermansia bacteria is lyophilized and the lyophilized bacteria retain the ability to prevent and / or treat reward system dysregulation.

[0066] As used herein, the term "extract" encompasses any component of the bacteria of the present invention, and in particular it encompasses both cellular and extracellular extracts that retain the ability to prevent and / or treat reward system dysregulation.

[0067] In practice, cell extracts include cytoplasmic extracts, membrane extracts, and combinations thereof, in particular extracts obtained from fractionation techniques. Cell extracts can be obtained by any standard chemical (SDS, proteinase K, lysozyme, combinations thereof, etc.) and / or mechanical (sonication, pressure) fractionation techniques, or adaptations thereof.

[0068] In practice, the extracellular extract may contain secretory fractions, particularly soluble compounds, or extracellular vesicles (EVs). As used herein, the term "extracellular vesicles" encompasses exosomes, exosome-like vesicles, microvesicles (or ectosomes) and apoptotic bodies. In some embodiments, the extracellular extract is an extracellular vesicle. In some embodiments, the extracellular extract is a secretory fraction. In some embodiments, the extracellular extract contains secretory molecules. In practice, the secretory fraction may be isolated and / or purified from the medium by any suitable method known in the state of the art or by a method adapted thereto. Exemplarily, the extracellular extract may be isolated from the medium by differential centrifugation; by polymer precipitation; by high performance liquid chromatography (HPLC), a combination thereof, and the like.

[0069] A non-limiting example of a method for differential centrifugation from culture medium includes the following steps: - a 10-20 min centrifugation step at a speed of about 300 × g to about 500 × g to remove cells; - a 10-20 min centrifugation step at a speed of about 1,500 × g to about 3,000 × g to remove dead cells; - a 20-45 min centrifugation step at a speed of about 7,500 × g to about 15,000 × g to remove necrotic tissue debris; -One or more ultracentrifugation steps at a speed of about 100,000 x g to about 200,000 x g for 30 to 120 min to pellet the exosomes.

[0070] Alternative methods for isolating exosomes may utilize commercially available kits, such as, for example, the exoEasy Maxi Kit (Qiagen®) or the Total Exosome Isolation Kit (Thermo Fisher Scientific®).

[0071] In practice, cellular and / or extracellular extracts may contain nucleic acids, proteins, carbohydrates, lipids, and combinations thereof, such as lipoproteins, glycolipids and glycoproteins, bacterial metabolites, organic acids, inorganic acids, bases, peptides, enzymes and coenzymes, amino acids, carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids, vitamins, biologically active compounds, metabolites, e.g., metabolites including inorganic components.

[0072] As used herein, the term "fragment of the bacterium of the invention" encompasses any part of the cell of the bacterium of the invention. Preferably, said fragment is a membrane fraction obtained by membrane preparation. Membrane preparations of microorganisms belonging to the genus Akkermansia can be obtained by methods known in the art. Alternatively, whole cell preparations are also envisaged. Preferably, the fragments described herein of the microorganism of the invention retain the ability to prevent and / or treat reward system dysregulation.

[0073] As used in the context of the present invention, the term "Akkermansia bacterium of the present invention" also encompasses derivatives or mutants or analogues of said bacterium which retain the ability to prevent and / or treat reward system regulation disorders.

[0074] It is understood that the reward system dysregulation according to the present invention can be diagnosed and / or monitored by evaluation of clinical symptoms with or without the aid of a dedicated questionnaire. In practice, the diagnosis and / or monitoring of the reward system dysregulation can be carried out by a certified operator.

[0075] According to certain embodiments, the reward system dysregulation is selected from the group including or consisting of psychiatric disorders, neurological disorders, disorders due to side effects of treatment, and combinations thereof.

[0076] In one embodiment, the reward system dysregulation is a psychiatric disorder. According to some embodiments, the psychiatric disorder is selected from the group including or consisting of addiction-related disorders, eating disorders, affective disorders, obsessive-compulsive disorders, schizophrenia, attention-deficit hyperactivity disorder (ADHD), autism spectrum disorders, anxiety disorders, and the like.

[0077] In one embodiment, the psychiatric disorder is an addiction disorder. According to some embodiments, the addiction-related disorder is selected from the group including or consisting of alcohol-related addiction, drug-related addiction, tobacco or nicotine addiction, gaming-related addiction, and the like.

[0078] In one embodiment, the mental disorder is an eating disorder or eating-related disorder. According to a particular embodiment, the eating disorder or eating-related disorder comprises or is selected from the group consisting of bulimia nervosa, binge eating disorder, anorexia nervosa, pica, rumination disorder, purging disorder, night eating syndrome, avoidance-restrictive food intake disorder, overweight-related disorder, and obesity-related disorder. Within the scope of the present invention, the terms "anorexia nervosa" and "anorexia" are used interchangeably. Within the scope of the present invention, the terms "bulimia nervosa" and "binge eating disorder" are used interchangeably. In one embodiment, anorexia nervosa includes restrictive type and binge eating / purging type.

[0079] According to certain embodiments, the eating disorder or eating related disorder includes or is selected from the group consisting of overweight related disorder, obesity related disorder, bulimia, anorexia, pica, rumination disorder, purging disorder, night eating syndrome, avoidance and restrictive food intake disorder, food craving, compulsive eating disorder, impulsive eating disorder, failure of calorie restricted diet, weight loss non-responder or non-responder to dietary advice for weight loss, etc. According to certain embodiments, the eating disorder or eating related disorder includes or is selected from the group consisting of overweight related disorder, obesity related disorder, bulimia, anorexia, pica, rumination disorder, purging disorder, night eating syndrome, and avoidance and restrictive food intake disorder. According to certain embodiments, the eating disorder or eating related disorder includes or is selected from the group consisting of overweight related disorder or obesity related disorder.

[0080] In some embodiments, eating disorders include food addiction, eating addiction, food cravings, food seeking, compulsive eating disorders, impulsive eating disorders, failure of a calorie restricted diet, weight loss non-responders or non-responders to dietary advice for weight loss.

[0081] In some embodiments, an eating disorder or eating-related disorder in the context of the present invention is a disorder associated with, associated with, or caused by dysregulated or abnormal processing of the food reward system. Thus, in one embodiment, the reward system dysregulation of the present invention is a food reward system dysregulation.

[0082] As used herein, an individual with an overweight-related disorder has a body mass index (BMI) of about 25.0 to about 29.9. As used herein, an individual with an obesity-related disorder has a body mass index (BMI) of greater than about 30.0.

[0083] In one embodiment, the individual has a body mass index (BMI) of about 25.0 to about 30.0, in another embodiment, the individual has a body mass index (BMI) of greater than about 30.0.

[0084] In one embodiment, the individual has a body mass index (BMI) of about 18.0 to about 25.0, in another embodiment, the individual has a body mass index (BMI) of greater than about 18.0.

[0085] In one embodiment, the eating related disorder is bulimia. In one embodiment, the eating related disorder is an overweight related disorder or an obesity related disorder. In one embodiment, the eating related disorder is an overweight related disorder. In one embodiment, the eating related disorder is an obesity related disorder. In one embodiment, the eating related disorder is a binge eating disorder. In one embodiment, the eating related disorder is anorexia. In one embodiment, the eating related disorder is pica. In one embodiment, the eating related disorder is a rumination disorder. In one embodiment, the eating related disorder is a purging disorder. In one embodiment, the eating related disorder is night eating syndrome. In one embodiment, the eating related disorder is avoidant-restrictive food intake disorder.

[0086] In one embodiment, the reward system dysregulation is a neurological disorder, hi certain embodiments, the neurological disorder is selected from the group including or consisting of Parkinson's disease, Tourette's syndrome, and the like.

[0087] In one embodiment, the reward system dysregulation is a disorder caused by a side effect of treatment. In certain embodiments, the disorder caused by a side effect of treatment is selected from the group consisting of or includes gaming addiction, shopping addiction, food addiction such as binge eating, hypersexuality, etc.

[0088] In some embodiments, the reward system dysregulation of the present invention is an eating disorder or an addictive disorder.

[0089] According to some embodiments, the composition is administered to an animal individual, preferably a mammalian individual, more preferably a human individual.

[0090] In one embodiment, the individual is a mammalian individual. In one embodiment, the individual is a human individual. In one embodiment, the individual is male. In one embodiment, the individual is female.

[0091] According to certain embodiments, the compositions are administered orally or rectally.

[0092] In one embodiment, the composition is administered to the gastrointestinal tract. It should be understood that the gastrointestinal tract is the final location of the bacteria according to the invention. In other words, the bacteria according to the invention are intended to be integrated into the microflora of an individual.

[0093] In one embodiment, the composition is a solid composition. In practice, solid forms adapted for oral administration include, but are not limited to, pills, tablets, capsules, soft gelatin capsules, hard gelatin capsules, dragees, granules, gums, chewing gums, caplets, compressed tablets, cachets, wafers, dragees, dragees, or dispersible / or disintegrating tablets, powders, solid forms suitable for solution or suspension in liquid prior to oral administration, and effervescent tablets.

[0094] In one embodiment, the composition is a liquid composition. Indeed, liquid forms adapted for oral administration include, but are not limited to, solutions, suspensions, drinking solutions, elixirs, sealed ampoules, quick-acting liquids, drenches, syrups, solutions and sprays.

[0095] According to some embodiments, the bacteria is about 1×10 2 CFU / g ~ approx. 1 x 10 12 CFU / g of composition, preferably about 1×10 3 CFU / g ~ approx. 1 x 10 11 CFU / g of composition, more preferably about 1×10 4 CFU / g ~ approx. 1 x 10 10 The bacteria is administered in a dosage containing about 1×10 CFU / g of the composition. In one embodiment, the bacteria is administered in a dosage containing about 1×10 4 CFU / g ~ approx. 1 x 10 11 CFU / g of composition, approximately 1 x 10 5 CFU / g ~ approx. 1 x 10 11 CFU / g of composition, approximately 1 x 10 6 CFU / g ~ approx. 1 x 10 11 CFU / g of composition, approximately 1 x 10 7 CFU / g ~ approx. 1 x 10 11 CFU / g of composition or approximately 1 x 10 8 CFU / g ~ approx. 1 x 10 11 The composition is administered in a dosage containing CFU / g of the composition.

[0096] As used herein, "CFU" means "colony forming unit." As used herein, "about 1 x 10 2 CFU / g ~ approx. 1 x 10 12 The term "CFU / g" means 1 × 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8, 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains CFU / g.

[0097] According to some embodiments, the bacteria is about 1×10 2 cells / g ~ approx. 1×10 12 As used herein, "about 1 x 10 2 cells / g ~ approx. 1×10 12 The term "cells / g" refers to 1 x 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains cells / g.

[0098] According to some embodiments, when the composition is a solid composition, the bacteria is present in an amount of about 1×10 2 CFU / g ~ approx. 1 x 10 12 CFU / g of the composition. As used herein, "about 1 x 10 2 CFU / g ~ approx. 1 x 10 12 The term "CFU / g" means 1 × 10 2 , 5×10 2 , 1×10 3 , 5×103 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains CFU / g.

[0099] According to some embodiments, when the composition is a solid composition, the bacteria is present in an amount of about 1×10 2 cells / g ~ approx. 1×10 12 As used herein, "about 1 x 10 2 cells / g ~ approx. 1×10 12 The term "cells / g" refers to 1 x 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains cells / g.

[0100] According to some embodiments, when the composition is a liquid composition, the bacteria is present in an amount of about 1×102 CFU / ml ~ approx. 1 x 10 12 CFU / ml of the composition. As used herein, "about 1 x 10 2 CFU / ml ~ approx. 1 x 10 12 The term "CFU / ml" means 1 × 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains CFU / ml.

[0101] According to some embodiments, when the composition is a liquid composition, the bacteria is present in an amount of about 1×10 2 cells / ml ~ approx. 1×10 12 As used herein, "about 1 x 10 2 cells / ml ~ approx. 1×10 12 The term "cells / ml" refers to 1 × 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×109 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 and 1 × 10 12 Contains cells / ml.

[0102] According to certain embodiments, the composition further comprises one or more additional active agents.

[0103] In one embodiment, the one or more additional active agents are therapeutic agents, hi another embodiment, the one or more additional active agents are nutritional agents.

[0104] According to certain embodiments, the one or more additional active agents are one or more beneficial microorganisms. In other words, in one embodiment, the composition further comprises one or more beneficial microorganisms.

[0105] According to some embodiments, the one or more beneficial microorganisms are selected from the group including or consisting of bacteria from the genera Verrucomicrobium, Clostridiaceae, Peptostreptococcus, Prevotellaceae, Methylobacteriaceae; Tannerellaceae, such as those from the genus Parabacteroides, Turicibacter, Coprococcus, Noelia, Prevotella, and Staphylococcus.

[0106] According to certain embodiments, the one or more additional active agents are one or more therapeutic agents known to prevent and / or treat the reward system dysregulation being treated.

[0107] According to certain embodiments, the composition is in the form of a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0108] In certain embodiments, pharma- ceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable oil saturated fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, wool fat, and the like, and any combination thereof.

[0109] According to certain embodiments, the composition is in the form of a nutritional composition further comprising a nutritionally acceptable carrier.

[0110] As used herein, the term "nutritional composition" is intended to refer to any food, food additive, health food, or fortified food, including liquid and solid foods. In practice, liquid foods include, but are not limited to, soups, soft drinks, sports drinks, energy drinks, fruit juices, lemonades, teas, milk-based drinks, and the like. In practice, solid foods include, but are not limited to, candy bars, cereal bars, energy bars, and the like. In one embodiment, the composition of the present invention is a food composition.

[0111] In some embodiments, the nutritional compositions of the present invention are for use in non-therapeutic applications or methods.

[0112] In some aspects, the present invention relates to a medicament comprising a therapeutically effective amount of one or more isolated bacteria of the genus Akkermansia and / or extracts and / or fragments thereof for use in the prevention and / or treatment of reward system dysregulation.

[0113] The present invention also relates to the use of a composition comprising one or more bacteria of the genus Akkermansia and / or an extract or fragment thereof for the manufacture of a medicament for the prevention and / or treatment of a reward system regulation disorder.

[0114] In some embodiments, the composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof is for use as an adjuvant for a therapeutic agent administered to a subject suffering from a reward system dysregulation disorder.

[0115] The present invention also relates to a medical device comprising, consisting of, or consisting essentially of one or more isolated bacteria of the genus Akkermansia and / or extracts and / or fragments thereof for use in preventing and / or treating reward system dysregulation. In one embodiment, the medical device according to the present invention comprises a therapeutically effective amount of one or more isolated bacteria of the genus Akkermansia and / or extracts and / or fragments thereof.

[0116] According to certain embodiments, the composition is included in a kit, which further comprises a means for administering said composition.

[0117] In some embodiments, the compositions, pharmaceutical compositions, nutritional compositions, medical devices or medicaments according to the present invention are sterile. In practice, methods for obtaining a sterile pharmaceutical composition include, but are not limited to, GMP synthesis (GMP stands for "Good Manufacturing Practice").

[0118] The present invention also relates to a method of restoring the reward system in a subject, the method comprising administering to said subject a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof.

[0119] As used herein, the term "reward system" relates to both the hedonic (or "pleasant") and motivational components of a behavior (eg, food intake).

[0120] In some embodiments, restoring the reward system restores motivation in a subject. In one embodiment, restoring the reward system restores motivation in a subject with abnormally reduced motivation. In another embodiment, restoring the reward system reduces motivation in a subject with abnormally increased motivation.

[0121] The present invention also relates to a method of modulating the dopaminergic system of the central nervous system in a subject in need thereof, comprising administering to said subject a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof in an amount effective to induce a reward response. In some embodiments, the reward response mimics a desired state in the subject.

[0122] The present invention also relates to a method of modulating the opioid system in a subject in need thereof, comprising administering to said subject a composition comprising one or more bacteria of the genus Akkermansia and / or their extracts and / or fragments in an amount effective to induce a reward response. In fact, the modulation of the opioid system has an effect on the "liking" component. In some embodiments, the reward response mimics a desired state in the subject. Without being bound by any theory, in certain embodiments, the effect of one or more bacteria of the genus Akkermansia and / or their extracts and / or fragments on the reward response modulates the opioid system.

[0123] The present invention also relates to a method for reducing expression of lipoprotein lipase (LPL) in the striatum in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof.

[0124] The present invention also relates to a method of reducing brain inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof.

[0125] The present invention also relates to a method for reducing pro-inflammatory factors, preferably pro-inflammatory cytokine release, and / or increasing anti-inflammatory factors, preferably anti-inflammatory cytokines, in a subject in need thereof, comprising administering to said subject an effective amount of a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof.

[0126] The present invention also relates to a method for improving the effectiveness of a treatment for a reward system dysregulation in a subject, comprising administering to the subject a composition comprising one or more bacteria of the genus Akkermansia and / or extracts and / or fragments thereof prior to, simultaneously with, or after the treatment for the reward system dysregulation.

[0127] Non-limiting examples of reward system dysregulation treatments include medications for addiction (eg, methadone for opioid addiction, disulfiram for alcoholism, or phentermine-topiramate for bulimia), behavioral or psychological counseling.

[0128] The present invention further relates to a method for restoring reward function in an individual in need thereof.In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia bacteria in the microbiota of a subject.In some embodiments, the composition is a prebiotic.In certain embodiments, the method comprises administering a composition comprising one or more Akkermansia bacteria and / or their extracts and / or fragments.In one embodiment, the method is non-therapeutic.

[0129] The present invention also relates to a method for reducing reward feeding in an individual in need thereof.In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia bacteria in microbiota.In certain embodiments, the method comprises administering a composition comprising one or more Akkermansia bacteria and / or their extracts and / or fragments.In one embodiment, the method is non-therapeutic.

[0130] The present invention further relates to a method for reducing the intake of palatable food in an individual in need thereof.In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia in microbiota.In a particular embodiment, the method comprises administering a composition comprising one or more bacteria of Akkermansia and / or their extracts and / or fragments.In one embodiment, the method is non-therapeutic.

[0131] The present invention further relates to a method for restoring motivation to eat in an individual in need thereof. In one embodiment, restoring motivation means enhancing or stimulating motivation in a subject with abnormally reduced motivation. In another embodiment, restoring motivation means reducing motivation in a subject with abnormally increased motivation. In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia in the microbiota. In a particular embodiment, the method comprises administering a composition comprising one or more bacteria of Akkermansia and / or extracts and / or fragments thereof. In one embodiment, the method is non-therapeutic.

[0132] The present invention further relates to a method for restoring appetite in a subject in need thereof. In one embodiment, restoring appetite means enhancing or improving appetite. In another embodiment, restoring appetite means reducing or inhibiting appetite. In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia in the microbiota. In a particular embodiment, the method comprises administering a composition comprising one or more bacteria of Akkermansia and / or extracts and / or fragments thereof. In one embodiment, the method is non-therapeutic.

[0133] The present invention further relates to a method for enhancing the "liking" response in an individual in need thereof. In one embodiment, the method comprises administering a composition comprising one or more bacteria of the genus Akkermansia and / or their extracts and / or fragments, in particular a composition comprising pasteurized Akkermansia bacteria. In one embodiment, the method comprises administering a composition comprising one or more active ingredients or substances that increase the level of Akkermansia in the microbiota. In one embodiment, the method is non-therapeutic. [Brief description of the drawings]

[0134] [Figure 1A-1D]A series of histograms and graphs showing that obesity is associated with alterations in the food reward system. (A) Food preference test showing intake in grams of HFHS and CT 3 hours after testing by lean and DIO mice. (B-C) Operant conditioning test showing active lever pressing during the breakpoint between four progressive ratio sessions (PR) and PR4 by lean and DIO mice. (D) Nucleus accumbens mRNA relative expression of dopamine receptor 2 (DRD2), dopamine receptor 1 (DRD1), tyrosine hydroxylase (TH) and dopamine transporter (DAT) in lean and DIO mice measured by real-time qPCR. Data are shown as mean ± SEM. P values ​​were obtained after 2-way ANOVA followed by Bonferroni post-hoc test (n=12 CT / 10 DIO) (A), repeated measures 2-way ANOVA followed by Bonferroni post-hoc test (n=12 mice / group) (B–C), unpaired Student's t-test or non-parametric Mann-Whitney test (n=9–10–12 mice / group) (B, C, D); *: p-value<0.05; ***: p-value<0.001; ****: p-value<0.0001 (between lean vs. DIO mice); $$$$: p-value<0.0001 (between CT vs. HFHS diet). [Fig. 2A-2F]A series of histograms showing that obesity is associated with inflammation in reward-related brain regions and blood-brain barrier alterations. (A) Nucleus accumbens mRNA relative expression of ionized calcium-binding adaptor (Iba1), glial fibrillary acidic protein (Gfap), cluster of differentiation 45 (Cd45), interleukin-β (Il1b), tumor necrosis factor-α (Tnfa) and interleukin-6 (Il6) in lean (black) and DIO (grey) mice (n=10 / group) as measured by real-time qPCR. (B-C) Representative immunofluorescence of the dorsal striatum (D.Str), ventral striatum (V.Str) whole, core and shell in lean (black) and DIO (grey) mice (n=4-5 / group), as well as quantification of the area occupied by both astrocytes (C) and GFAP+ cells (B) in these regions. (D-E) Representative immunofluorescence of the dorsal striatum (D.Str), ventral striatum (V.Str) whole, core and shell in lean (black) and DIO (grey) mice (n=5 mice / group), and quantification of both the area occupied by microglial cells (E) and Iba1+ cells (D) in these regions. (F) Ventral striatal mRNA relative expression of claudin-1 (Cldn1), claudin-5 (Cldn5), junction 1 (Zo1) and occludin (Ocln) in lean (black) and DIO (grey) mice (n=10 mice / group) measured by real-time qPCR. Data are shown as mean ± SEM. P values ​​were obtained after unpaired Student's t-test or nonparametric Mann-Whitney test; *: p value < 0.05; **: p value < 0.01; ***: p value < 0.001 (between Lean vs. DIO mice). [Figure 3A-3D]A series of histograms detailing immunofluorescence quantification. (A-B) Immunofluorescence quantification of the area occupied by astrocytes (A) and Gfap+ cells (B) in the right and left dorsal striatum (D.Str), whole ventral striatum (V.Str whole), ventral striatal core (V.Str core) and ventral striatal shell (V.Str shell) of lean (black) and DIO (grey) mice (n=4-5 mice / group). (C-D) Immunofluorescence quantification of the area occupied by microglial cells (C) and Iba1+ cells (D) in the right and left dorsal striatum (D.Str), whole ventral striatum (V.Str whole), ventral striatal core (V.Str core) and ventral striatal shell (V.Str shell) of lean (black) and DIO mice (grey) (n=5 mice / group). Data are shown as mean ± SEM. P values ​​were obtained after unpaired Student's t-test or nonparametric Mann-Whitney test; *: p value < 0.05; **: p value < 0.01 (between Lean vs. DIO mice). [Figure 4A-4B] A series of histograms and graphs showing that A. muciniphila administration restores the motivational component of food reward associated with obesity. Operant conditioning test showing active lever pressing during the four progressive ratio sessions (PR) and the breakpoint during PR4 (A) and maximum number of pellets acquired (B) by DIO mice treated with placebo (DIO_placebo) and A. muciniphila (DIO_Akk). Data are shown as mean ± SEM. P values ​​were obtained after repeated measures two-way ANOVA followed by Bonferroni post-hoc test (n = 6 mice / group) or unpaired Student's t-test or non-parametric Mann-Whitney test (n = 6 mice / group). *: p value < 0.05; **: p value < 0.01 (between DIO_placebo vs. DIO_Akk). $: p value < 0.05 (between CT vs. HFHS diet intake). [Figure 5A-5C]A series of histograms showing that Akkermansia muciniphila administration reduces markers in reward-related area inflammation associated with systemic and adiposity as well as striatal expression of LPL. (A) Relative striatal mRNA expression of ionized calcium-binding adaptor-1 (IBA-1), Toll-like receptor 4 (TLR4), glial fibrillary acidic protein (GFAP), and cluster of differentiation 45 (CD45) in placebo (DIO_placebo) and A. muciniphila (DIO_Akk)-treated DIO mice (n=5–8–10 / group) as measured by real-time qPCR. (B) Plasma concentration of the cytokine tumor necrosis factor alpha (TNFα). (C) Relative striatal mRNA expression of lipoprotein lipase (LPL) in placebo (DIO_placebo) and A. muciniphila (DIO_Akk)-treated DIO mice (n=8–10 / group) as measured by real-time qPCR. Data are shown as mean ± SEM. P values ​​were obtained after unpaired Student's t-test or nonparametric Mann-Whitney test; *: p value < 0.05; **: p value < 0.01 (between DIO_placebo vs. DIO_Akk). [Figure 6A-6B]A series of histograms showing that pasteurized A. muciniphila administration restores the preference component of food reward associated with obesity. (A) Food preference test showing the intake in grams of HFHS and CT by DIO mice treated with placebo (DIO_placebo) and pasteurized A. muciniphila (DIO_Akkpast) (A) and preference in percentage to HFHS 3 h after the test (B). The percentage of food preference was calculated based on HFHS intake (g) during the food preference test divided by the total food intake (g) ingested during the food preference test. Data are shown as mean ± SEM. p values ​​were obtained after 2-way ANOVA followed by Bonferroni post-hoc test (n = 6–8 mice / group). **: P value < 0.01 between DIO_placebo and DIO_Akkpast HFHS intake. $$$$: P value < 0.0001 (between CT vs. HFHS food intake) (A). p values ​​were obtained after Student's t test (n = 7-8 animals / group). ****: P value < 0; 0.0001 (between DIO_placebo and DIO_Akkpast) (B). [Figure 7A-7C] A series of graphs showing that pasteurized A. muciniphila administration improves the appetitive component of food reward associated with obesity. Operant conditioning test showing active lever pressing during four progressive ratio sessions (PR) (A), active lever pressing during four progressive ratio sessions (PR) (B), and maximum number of pellets acquired (C) by DIO mice treated with placebo (DIO_placebo) and pasteurized A. muciniphila (DIO_Akkpast). Data are shown as mean ± SEM. P values ​​were obtained after repeated measures two-way ANOVA followed by Bonferroni post-hoc test (n = 6–7 mice / group) (A) or Mann-Whitney test (n = 6–8 mice / group) (B, C). *: p value < 0.05 (between DIO_placebo and DIO_Akkpast). [Figure 8]Histogram showing that pasteurized A. muciniphila administration does not affect the expression of inflammatory markers in the striatum. Relative expression of striatal mRNA for ionized calcium-binding adaptor (Iba1), glial fibrillary acidic protein (Gfap), interleukin 6 (Il6), interleukin 1 beta (Il1b), tumor necrosis factor alpha (Tnfa) and Toll-like receptor 4 (Tlr4) in DIO mice treated with placebo (DIO_placebo) and A. muciniphila (DIO_Akkpast) as measured by real-time qPCR. Data are shown as mean ± SEM. P values ​​were obtained after Student's t-test or Mann-Whitney test (n=7–8 animals / group). [Figure 9A-B] A series of histograms showing that pasteurized Akkermansia muciniphila administration increases the area occupied by microglial cells in the striatum. Representative immunofluorescence of the whole, core and shell of the dorsal striatum (D.Str), ventral striatum (V.Str) of DIO_placebo and DIO_Akkpast mice (n=4–6 per group), as well as the number of Iba1+ cells in these regions (A) and the quantification of the area occupied by microglial cells (B). Data are shown as mean ± SEM. P values ​​were obtained after Student's t-test (between DIO_placebo and DIO_Akkpast). *: p value < 0.05 (between DIO_placebo and DIO_Akkpast). [Figure 10] Histogram showing that pasteurized A. muciniphila administration modulates the expression of markers of the M2 anti-inflammatory phenotype of microglial cells in the striatum. Relative expression of striatal mRNA for cluster of differentiation 11b (CD11b), cluster of differentiation 206 (Cd206) and arginase 1 (Arg1) in DIO mice treated with placebo (DIO_placebo) and pasteurized A. muciniphila (DIO_Akkpast) as measured by real-time qPCR. Data are shown as mean ± SEM. p values ​​were obtained after Student's t-test or Mann-Whitney test (n = 7–8 mice / group). [Figure 11]Histogram showing that pasteurized A. muciniphila administration reduces the expression of striatal LPL. Relative expression of striatal mRNA for lipoprotein lipase (LPl) in DIO mice treated with placebo (DIO_placebo) and pasteurized A. muciniphila (DIO_Akkpast) as measured by real-time qPCR. Data are shown as mean ± SEM. p values ​​were obtained after Student's t-test (n=7-8 mice / group). EXAMPLES

[0135] Working Example The present invention is further illustrated by the following examples.

[0136] Example 1: Diet-induced obese mice exhibit behavioral and neurological changes in response to food reward. material and method Mice and experimental design: All mouse experiments were approved by the ethical committee of UCLouvain, Universite catholique de Louvain, Health Sector of animal care under the specific number 2017 / UCL / MD / 005, and were performed according to the local ethical committee guidelines and in accordance with the Belgian law of 29 May 2013 for the protection of experimental animals. Body weight, food and water intake were recorded once a week. Body composition was assessed using a 7.5 MHz time-domain nuclear magnetic resonance (TD-NMR, LF50 Minispec, Bruker, Rheinstetten, Germany). Cohorts of 9-week-old specific opportunistic pathogen and specific pathogen-free (SOPF) male C57BL / 6J mice (Janvier laboratories, France) were housed in groups of 2 mice per cage in a controlled environment (room temperature 22 ± 2 °C, 12-h daylight cycle) and had free access to sterile food (irradiated food) and sterile water. Upon receipt, mice were allowed to acclimate for 1 week during which they were fed a control diet (CT, AIN93Mi, Research Diet, New Brunswick, NJ, USA). They were then randomly divided into two groups (40 mice, n=20 / group) and fed a control low-fat diet (CT, AIN93Mi) or a high-fat diet (HFD, 60% fat and 20% carbohydrate (kcal / 100g) D12492i, Research Diet, New Brunswick, NJ, USA) for 8 weeks. After 4 weeks of follow-up, mice were placed in behavioral cages and subjected to food preference and operant wall tests. During this last test, mice were food restricted and body weight was maintained at 85% of the initial weight (before behavioral testing) as previously described (de Wouters d'Oplinter A et al, Gut Microbes, 2021). Caloric restriction allowed for an enhancement of the reward response to stimuli.

[0137] Food preference testing: Mice were fed two diets: a low-fat, control diet (CT, AIN93Mi, Research Diet, New Brunswick, NJ, USA) or a high-fat, high-sucrose diet (HFHS, 45% fat and 27.8% sucrose (kcal / 100 g) D17110301i, Research Diet, New Brunswick, NJ, USA) in Phenotyper chambers (Noldus, The Netherlands) during a 3-h daytime period. Food intake was recorded in the well-fed state (with free access to food before and after testing) during a 3-h session at the end of the light phase.

[0138] Operant wall test: Appetitive component, related to the motivation to obtain reward, is assessed by the operant wall test as previously described (Cansell C et al, Molecular psychiatry, 2014) with some modifications. Each test session was performed at the end of the light phase in operant conditioning chambers (Phenotyper chambers, Noldus, The Netherlands) and analyzed by the provided software (Ethovision XT 14). Mice had intermittent access to the operant wall in their home cage. The operant wall system consists of two levers and two lights and a pellet dispenser. One lever was arbitrarily designated as active, meaning that pressing this lever initiates the delivery of sucrose pellets (5-TUT peanut butter flavored sucrose pellets, TestDiet, St. Louis, MO), and is associated with the light being turned on. On the other hand, the other lever, associated with the light being turned off, was arbitrarily designated as inactive and does not deliver reward. Mice were trained on the system overnight on a fixed ration schedule (one lever press corresponds to one reward) followed by two 2-h sessions. Mice were then transferred to a progressive ratio session (1 h 30 min) in which the number of lever presses required to obtain a reward was gradually increased per pellet (n+3).

[0139] Tissue Sampling: At the end of each experiment, mice were maintained under caloric restriction and exposed to HFHS for 1 h before anesthesia with isoflurane (Forene, Abbott, England). The purpose was to mimic the conditions of behavioral testing and to induce the reward system. Mice were then anesthetized with isoflurane and euthanized by exsanguination and cervical dislocation. Blood was collected from the portal vein and vena cava. The striatum and nucleus accumbens were precisely dissected and immediately immersed in liquid nitrogen before being stored at -80°C for further analysis.

[0140] RNA preparation and real-time qPCR analysis: Total RNA was extracted from the striatum using TriPure reagent (Roche). cDNA was prepared by reverse transcription of 1 μg of total RNA using the GoScript Reverse Transcriptase kit (Promega, Madison, WI, USA). Real-time PCR was performed on a QuantStudio 3 real-time PCR system (Thermo Fisher, Waltham, MA, USA). Rpl19 RNA was selected as a housekeeping gene. All samples were run in duplicate and data were analyzed according to the 2-ΔΔCT method. The identity and purity of the amplified products were assessed by melting curve analysis at the end of the amplification.

[0141] Statistical analysis: Statistical analysis was performed using GraphPad Prism version 9.1.2 for windows (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± SEM. Differences between two groups were evaluated using unpaired Student's t-test. If variances were significantly different between groups by Fisher's test, nonparametric (Mann-Whitney) tests were performed. Differences between two groups and between different time points were evaluated using Bonferroni post-hoc tests following two-way ANOVA repeated measures.

[0142] result Nine-week-old lean or diet-induced obese mice were followed for nine weeks on a control low-fat diet (CT, lean group) or a high-fat diet (HFD). After four weeks on each diet to establish obesity, the preference and appetitive components of the food reward system were assessed in behavioral tests.

[0143] The preference or hedonic component of food intake was assessed during food preference testing by first analyzing the preference for the palatable diet (HFHS, high fat and high sucrose) compared to the control diet (CT). Mice were exposed to both diets in a well-satiated state (with free access to diet before and after testing) during a 3-h session at the end of the light phase. In this situation, HFHS preference should suggest a rewarding response specifically towards the palatable diet. As expected and consistent with the literature (Delbes AS et al., Front Endocrinol, 2018; Carlin J et al., Obesity, 2013), lean mice showed greater preference for the palatable diet (HFHS) compared to the control diet, as indicated by higher intake of HFHS compared to the intake of CT diet (p<0.0001 between CT vs. HFHS in lean group, Fig. 1A); in contrast, obese mice (DIO) showed no preference for HFHS compared to the CT diet (p=0.77 between CT vs. HFHS in DIO group, Fig. 1A). Importantly, DIO mice consumed more than 2-fold less HFHS than lean mice (1.16 g vs. 0.57 g between lean and DIO mice, respectively, p<0.0001). Since this test represents the first exposure to a palatable meal, which can stimulate the mesolimbic system and induce enjoyment, the absence of preference for the palatable meal in obese mice, and reduced intake of this meal, suggests a dysregulation of the hedonic component of food intake associated with obesity.

[0144] Next, the motivational component of food reward was assessed by subjecting mice to an operant conditioning task in which their desire to obtain rewarding food (peanut butter flavored sucrose pellets) was tested. Lean and DIO mice were first assessed for incentive motivation on a fixed ratio schedule: one lever press delivered one sucrose pellet. After four sessions with fixed ratio (FR), mice were transferred to progressive ratio sessions (PR), which required an increasing number of lever presses [three more lever presses for each subsequent reinforcer (r=3n+3; n=number of reinforcers)] to obtain a novel sucrose pellet. The PR sessions thereby measure the amount of effort the animals were willing to make to obtain a food reward, which depends on the motivational aspect of the reward system. Compared to lean mice, DIO mice pressed significantly less levers to obtain a reward during PR sessions, suggesting reduced motivation (p<0.0001 between lean and DIO mice, Fig. 1B-C). Consistent with the number of lever presses, the breakpoints or the number of sucrose pellets acquired during the session were significantly lower in the DIO group than in the lean group (p=0.0001 and p<0.0001 between lean and DIO mice in PR1 and PR2-4, respectively, Figure 1B-C). These results indicate a dysregulation of the motivational components of the reward system associated with obesity, as previously described (Delbes AS et al., Front Endocrinol, 2018).

[0145] Given the observed dysregulation of the reward system in terms of behavior in obese animals compared to lean mice, the reward system was further investigated by analyzing the dopaminergic system in mesocorticolimbic structures of the brain (Figure 1D). Expression of dopaminergic receptors 2 (Drd2) and 1 (Drd1) was decreased in the ventral striatum of obese mice (p=0.028 and p=0.050, respectively), whereas dopamine transporter (DAT), involved in the recapture of dopamine, tended to be increased (p=0.068; Figure 1D). Expression of tyrosine hydroxylase (Th), the rate-limiting enzyme synthesizing dopamine, was not affected in DIO mice.

[0146] The altered expression of key dopaminergic markers associated with obesity indicates a downregulation of dopamine pathways and reflects the behavioral dysregulation of the liking and appetitive components of food reward observed during the food preference and operant wall tests, respectively. Collectively, these results indicate an alteration of the reward system in obese mice for behavioral and neural approaches, as previously described in the literature (de Wouters d'Oplinter A et al., Gut Microbes, 2021).

[0147] Example 2: Obesity is associated with inflammation in reward-related brain regions and alterations in the blood-brain barrier. material and method Mouse experiments, tissue sampling, RNA preparation, real-time qPCR and statistical analysis: see Example 1. Immunofluorescence: At the end of the experiment (see Example 1), mice were anesthetized with isoflurane and perfused transcardially using a solution of cold phosphate-buffered saline (PBS) followed by cold 4% (w / v) paraformaldehyde (PFA) solution. Whole brains were carefully harvested and post-fixed overnight at 4°C in 4% PFA, cryoprotected overnight at 4°C in a solution of sucrose 30% (w / v), and subsequently frozen in cold isopentane and stored at -80°C, as previously described (Everard A et al. Nat Commun 2019). Serial coronal cryosections 20 μm thick of fixed brains were mounted on SuperFrost Plus slides (Menzel Glaser) and kept at -20°C. For the nucleus accumbens and striatum, eight serial sections per animal were taken from 0.61 mm to 1.41 mm from the bregma according to stereotaxic coordinates of mouse brain (Paxinos, Franklin). Immunofluorescence was performed using the tyramide signal amplification (TSA) technique as previously described (Everard A et al., Nat Commun 2019). Briefly, after antigen retrieval (Dako S1699) by heating (2100 Antigen Retriever (Aptum)), endogenous peroxidase was inhibited in MeOH solution containing 0.1% (v / v) H2O2. Sections were then incubated in blocking solution (TBS, 5% BSA, 0.1% Tween 20) for 45 min, followed by overnight incubation with primary antibodies (anti-GFAP 1 / 10,000, ab5804 (Merck) or anti-Iba-1 1 / 500, PA5-27436 (ThermoFischer)). After washing, sections were incubated with horseradish peroxidase-labeled secondary antibodies (DAKO K4003) for 1 h. Fluorescent signals were amplified using Alexa Fluor488 tyramide reagent (B40953 (ThermoFischer)). Finally, nuclei were stained with Hoechst 33342 (H1399 Invitrogen). Slides were dehydrated and mounted with Dako Fluorescence Mounting Medium.Fluorescent GFAP scans were obtained using an Oyster scanner (3DHistech Pannoramic P250 Flash III) and fluorescent Iba-1 scans were obtained using a Zeiss scanner (Axioscan.z1). After a blinding procedure, regions of interest (ROIs) corresponding to the nucleus accumbens core, shell and dorsal striatum were delimited on each section using the mouse brain stereotaxic coordinates (Paxinos, Flanklin) as reference and the % of green area was measured using Fiji software48. Positive neurons within each ROI were counted manually and the average value was obtained for each animal. At least three brain sections per animal were considered.

[0148] result It has been widely described that obesity is associated with low-grade inflammation in several tissues, including the brain (Guillemot-Legris O et al., Trends Neurosci, 2017). Importantly, so-called neuroinflammation has been reported to alter brain function (Decarie-Spain L et al., Brain Behav Immun Health, 2021). It is therefore hypothesized that dysregulation of dopaminergic pathways during obesity may be related to inflammation in reward-related brain regions. Since brain inflammation is associated with microglial and astroglial activation, as reflected by increased expression of ionized calcium-binding adaptor protein-1 (Iba1) and glial fibrillary acidic protein (GFAP), respectively, we analyzed the expression of the receptor for LPS (Tlr4), the infiltrating immune cell marker (classification of surface antigen 45) (Cd45)) and pro-inflammatory cytokines (interleukin-6 (Il6), interleukin-1β (Il1b) as well as tumor necrosis factor alpha).

[0149] A significant increase in the expression of CD45, IL-1β (p=0.044 and p=0.025, respectively), and a trend towards increased TNFα (p=0.0524) expression were detected in obese mice compared to lean mice (Figure 2A), suggesting the induction of neuroinflammation in mesocorticolimbic regions by high-fat diet-induced obesity. Immunohistochemical staining of GFAP and Iba1 in the striatum was performed in reward-related regions. DIO induced astroglial activation in the dorsal striatum and ventral striatal core, as shown by increased staining of the labeled areas in the DIO group compared to the lean group (p=0.049 and 0.051, respectively (comparison between lean and DIO mice, Figure 2B-C)). Strikingly, astrocytes were mainly activated in the right dorsal and ventral striatum, whereas these regions in the left hemisphere did not show astroglial activation (Figure 3A-D). Iba1 immunostaining did not reveal any differences in microglial cell activation (Figures 2D-E).

[0150] The blood-brain barrier (BBB) ​​is essential to protect brain structures from toxins, pathogens and excessive immune cell infiltration, and to maintain neuronal integrity. Since neuroinflammation is associated with BBB disruption in several neurological disorders, including some associated with obesity, we further analyzed the integrity of the BBB in reward brain regions by measuring the expression of key tight junction proteins. Interestingly, the expression levels of claudin-5 (Cldn5) and junction 1 (Zo1) were significantly decreased in obese mice compared to lean mice (p=0.0033 and p=0.0004, respectively, between lean and DIO mice), whereas claudin-1 (Cldn1) and occludin (Ocln) levels were unchanged (Figure 2F).

[0151] This series of results shows for the first time that behavioral and neuronal dysfunction of the reward system in obesity is associated with markers of neuroinflammation and BBB alterations.

[0152] Example 3: Administration of Akkermansia muciniphila restores the motivational component of food reward altered by DIO. The gut microbiota plays an important role in systemic inflammation during obesity. Indeed, a long-term high-fat diet is associated with changes in gut microbiota composition, as well as increased translocation of the bacterial component lipopolysaccharide (LPS) through the intestinal barrier. This increase in plasma LPS, also called metabolic endotoxemia, generates low-grade inflammation through the activation of TLR4. Saturated fatty acids also activate TLR4 and enhance inflammation.

[0153] In this context, we identified Akkermansia muciniphila (A. muciniphila) as a beneficial bacterium that counteracts metabolic disorders including diet-induced obesity and low-grade inflammation (Everard A et al., Proc Natl Acad Sci USA, 2013). Therefore, we evaluated the potential impact of A. muciniphila administration on the reward system in obese animals.

[0154] material and method Operant wall testing and statistical analysis: See Example 1.

[0155] Mouse experimental design: Cohorts of 9-week-old specific opportunistic pathogen and specific pathogen free (SOPF) male C57BL / 6J mice (Janvier laboratories, France) were housed in a controlled environment (room temperature 22 ± 2 °C, 12-h light cycle) in groups of 2 mice per cage with free access to sterile food (irradiated food) and sterile water. Upon receipt, mice were allowed to acclimate for 1 week during which they were fed a control diet (CT,AIN93Mi, Research Diet, New Brunswick, NJ, USA). They were then randomly divided into two groups (20 mice, n = 10 / group) and fed a high-fat diet (HFD, 60% fat and 20% carbohydrate (kcal / 100 g) D12492i, Research Diet, New Brunswick, NJ, USA) for 8 weeks. One group contained 2 × 10 8Mice were treated with Akkermansia muciniphila by oral gavage at a dose of 1000 CFU. A placebo group was orally administered an equivalent volume of sterile anoxic PBS containing a similar final concentration of glycerol (2.5% volume / volume). Treatment continued until the end of the experiment, which lasted 8 weeks in total. After 4 weeks of follow-up, mice were placed in behavioral cages and subjected to the operant wall test. During this last test, mice were food restricted and body weight was maintained at 85% of the initial weight (before behavioral testing) as previously described (de Wouters d'Oplinter A et al, Gut Microbes, 2021). Caloric restriction allows for an enhancement of the reward response to stimuli.

[0156] result The food reward system was investigated by investigating motivation using an operant conditioning task. Progressive ratio sessions (PR), which require an increasing number of lever presses to obtain a novel food reward, showed a statistically significant increase in motivation in obese mice treated with A. muciniphila compared to placebo-treated obese mice at PR4 (p=0.0022 (comparison between DIO_placebo and DIO_Akk groups), Figure 4A-B). A. muciniphila-treated mice reached a similar number of lever presses as lean mice during their first progressive ratio session (see Figure 1B, mean=666 (Lean) vs. Figure 3A, mean=638 (DIO_Akk)). Consistent with the lever pressing parameters, the maximum number of reinforcers acquired tended to be increased in obese mice treated with A. muciniphila compared to placebo-treated mice (p=0.075, Figure 4B).

[0157] Considering the greater number of lever presses to obtain more sucrose pellets than obese placebo mice, these results indicate that administration of A. muciniphila can restore the motivational changes induced by a high-fat diet.

[0158] Example 4: The beneficial effects of Akkermansia muciniphila on the reward system imply a reduction in systemic and neuroinflammation as well as a reduction in striatal LPl expression. From a mechanism of action perspective, the dysregulation of the reward system associated with obesity may be due to inflammation in reward-related brain regions. Based on the ability of A. muciniphila to reverse the changes in motivation induced by a high-fat diet (Figure 4A-B), and because a systemic anti-inflammatory effect has been previously reported for this bacterium, we assessed inflammation in reward-related brain regions in obese mice treated or not with A. muciniphila.

[0159] material and method Mouse experimental design: See Example 3.

[0160] RNA preparation, real-time qPCR and statistical analysis: see Example 1.

[0161] Plasma multiplex analysis: Plasma concentrations of TNFα were measured using a multiplex assay kit based on chemiluminescence detection according to the manufacturer's instructions. The analysis was performed using a QuickPlex SQ 120 instrument (MSD) and DISCOVERY WORKBENCH® 4.0 software.

[0162] result We found that administration of A. muciniphila reduced plasma levels of TNFα (p=0.040 (comparison of DIO_placebo and DIO_Akk mice), Figure 5B). Consistent with this reduction in the levels of systemic inflammatory markers, expression of TLR4 was substantially reduced in the striatum of A. muciniphila-treated mice compared to placebo-treated mice (p=0.0015 (comparison of DIO_placebo and DIO_Akk), Figure 5A). Striatal expression of a marker of infiltrating immune cells (Cd45) also tended to be reduced in A. muciniphila-treated mice compared to placebo-treated mice (p=0.072 (comparison of DIO_placebo and DIO_Akk), Figure 5A). However, striatal expression of a marker of microglia (Iba1) and astrocytes (Gfap) was unchanged between the DIO group compared to the DIO_Akk group (Figure 5A). Collectively, our results suggest that A. muciniphila supplementation reverses the motivational changes associated with obesity, possibly via modulation of Tlr4 expression and immune cell infiltration in mesocorticolimbic structures.

[0163] According to previous studies, a potential mechanism linking changes in behavioral reward with dopaminergic transmission involves central lipid sensing via lipid processing LPL. Similar increases in motivational performance on food-seeking behavior have been assessed with viral-mediated knockdown of LPL in mice in a progressive ratio operant conditioning paradigm (Berland C et al., Cell Metab, 2020; Cansell C et al, Mol Psychiatry, 2014). Importantly, in our cohort, mice administered A. muciniphila showed a highly significant reduction in LPL expression in the striatum compared to placebo-treated obese mice (p=0.0058, Figure 5C).

[0164] A. muciniphila supplementation is an additional and innovative approach to restore food reward behavior during obesity. These results pave the way for further application of A. muciniphila in improving behavior in other neuropsychiatric disorders such as Parkinson's disease or Alzheimer's disease, which are associated with CNS inflammation.

[0165] Example 5: Administration of pasteurized Akkermansia muciniphila restores hedonic / liking of food rewards altered by DIO and tends to improve motivational components.

[0166] material and method Food preference tests, operant wall tests and statistical analyses: see Example 1.

[0167] Mouse experimental design: Cohorts of 9-week-old specific opportunistic pathogen and specific pathogen free (SOPF) male C57BL / 6J mice (Janvier laboratories, France) were housed in groups of 2 mice per cage in a controlled environment (22±2°C room temperature, 12-h light cycle) with free access to sterile food (irradiated food) and sterile water. Upon receipt, mice were allowed to acclimate for 1 week during which they were fed a control diet (CT,AIN93Mi, Research Diet, New Brunswick, NJ, USA). They were then randomly divided into two groups (32 mice, n=16 / group) and fed a high-fat diet (HFD, 60% fat and 20% carbohydrate (kcal / 100g) D12492i, Research Diet, New Brunswick, NJ, USA) for 8 weeks. One group of HFD contained 2 × 10 cells suspended in sterile, oxygen-free PBS (DIO_Akkpast) (pasteurized at 70 °C for 30 min) as previously described (Plovier et al., Nat Med, 2017). 8Mice were treated by oral gavage with a dose of 1000 CFU of pasteurized Akkermansia muciniphila. The last group of HFD mice (DIO_placebo) was orally administered an equivalent volume of sterile anoxic PBS containing a similar final concentration of glycerol (2.5% volume / volume). Treatment continued until the end of the experiment, which lasted a total of 8 weeks. After 3 weeks of follow-up, mice were placed in behavioral cages and food preference and operant wall tests were performed. During this last test, mice were food restricted and body weight was maintained at 85% of the initial weight (before this behavioral test) as previously described (de Wouters d'Oplinter A et al, Gut Microbes, 2021). Caloric restriction allows for an enhancement of the reward response to stimuli.

[0168] result In this study, we first performed a food preference test to evaluate the preference component of food reward. As expected, DIO_placebo mice did not show any preference for the palatable (HFHS) since they consumed the same amount of HFHS as the control diet, corroborating the change in preference component associated with obesity (Figure 6). Importantly, the results show that administration of pasteurized A. muciniphila increases hedonic food intake since pasteurized A. muciniphila-supplemented mice (DIO_Akkpast) consumed more HFHS than placebo-treated mice (DIO_placebo) (Figure 6A). Furthermore, pasteurized A. muciniphila supplementation restores preference for the HFHS diet compared to the CT diet (Figure 6B). These results indicate that pasteurized A. muciniphila supplementation has a beneficial effect on the preference component of food intake in the context of obesity since it counteracts high-fat diet-induced changes. Thus, pasteurized A. muciniphila restores the palatable component of the food reward.

[0169] The motivation of the mice for food reward was also evaluated by the operant wall test. Interestingly, mice treated with pasteurized A. muciniphila (DIO_Akkpast) pressed the active lever more than placebo-treated mice (DIO) (Figure 7A-B).

[0170] Consistent with the lever-pressing parameters, the maximum number of reinforcers acquired tends to be increased in obese mice treated with pasteurized A. muciniphila compared to placebo-treated mice (Figure 7C). Thus, pasteurized A. muciniphila improves the appetitive component of the food reward.

[0171] Taken together, the studies clearly demonstrate the beneficial effects of pasteurized A. muciniphila on behavioral patterns of the food reward system.

[0172] Example 6: Pasteurized Akkermansia muciniphila modulates the inflammatory profile of microglial cells in the striatum during obesity.

[0173] material and method Mouse experimental design: See Example 5.

[0174] RNA preparation, real-time qPCR, immunofluorescence and statistical analysis: see Example 1.

[0175] result We next investigated the effect of pasteurized A. muciniphila on inflammation and LPI expression in reward-related brain regions. We first evaluated the expression of inflammatory markers in the striatum of HFD and HFD mice treated with pasteurized A. muciniphila. Upon pasteurization, A. muciniphila did not appear to affect pro-inflammatory markers in reward-related brain regions, since there were no significant differences in the expression of Iba1 (microglial activation); Gfap (astroglial cells); IL6, Il1b, and Tnfα (pro-inflammatory cytokines); and Tlr4 (receptor for LPS and some fatty acids) between placebo and A. muciniphila-supplemented mice (Figure 8).

[0176] To investigate and visualize inflammation more deeply, immunohistochemical staining for microglial cell (immune cell in the brain) marker (Iba1) in reward-related regions was performed. Surprisingly, it was found that administration of pasteurized A. muciniphila induced an increase in the number of microglial cells in the dorsal striatum and ventral striatal core, as indicated by increased staining of Iba1+ cells in the DIO_Akkpast group compared to the lean group (Figure 9A). Consistently, as the number of microglial cells increases in these reward-related brain regions, an increase in the Iba1 area occupied in these regions in the DIO_Akkpast group was observed as well (Figure 9B).

[0177] Importantly, activation of microglial cells (by increasing their number or size) can induce either a pro- or anti-inflammatory phenotype. Erny et al. have demonstrated that the gut microbiota can modulate microglial maturation and function, so they further investigated the effect of pasteurized A. muciniphila on the activity of the anti-inflammatory microglial phenotype (Erny et al., Nat Neurosci, 2015). Clusters of differentiation 11b and 206 (Cd11b and Cd206) and arginase 1 (Arg1) are often used as markers of this anti-inflammatory microglial phenotype (Rossi et al., Cell Death Dis, 2018).

[0178] Interestingly, we found a trend towards an increase in two distinct inflammatory markers, Cd206 and Arg1, in the striatum of the DIO_Akkpast compared to the DIO_placebo group (Figure 10). These data suggest that the increase and activation of microglial cells by pasteurized A. muciniphila is associated with an anti-inflammatory profile.

[0179] Example 7: The beneficial effects of Akkermansia muciniphila on the reward system imply a reduction in striatal LPl.

[0180] material and method Mouse experimental design: See Example 5.

[0181] Mouse preparation, real-time qPCR and statistical analysis: see Example 1.

[0182] result Mice administered A. muciniphila showed a significant decrease in LPl expression in the striatum compared to placebo-treated obese mice (p=0.0456 after Student's t-test between DIO_placebo and DIO_Akk_Past, FIG. 11).

[0183] References Avena NMand Bocarsly ME,Dysregulation of Brain Reward Systems in Eating Disorders:Neurochemical Information from Animal Models of Binge Eating,Bulimia Nervosa,and Anorexia Nervosa.Neuropharmacology 2012;63(1):87-96. Berland C, Cansell C, Hnasko TS, Magnan C, Luquet S. Dietary triglycerides as signaling molecules that reward influence and motivation. Curr Opin Behav Sci 2016;9:126-35. Berland C, Montalban E, Perrin E, Di Miceli M, Nakamura Y, Martinat M, et al.Circulating Triglycerides Gate Dopamine-Associated Behaviors through DRD2-Expressing Neurons.Cell Metab 2020;31:773-90 e11. Cani PD,Amar J,Iglesias MA,Poggi M,Knauf C,Bastelica D,et al.Metabolic endotoxemia initiates obesity and insulin resistance.Diabetes 2007;56:1761-72. Cani PD,Van Hul M,Lefort C,Depommier C,Rastelli M,Everard A.Microbial regulation of organismal energy homeostasis.Nat Metab 2019;1:34-46. Cansell C,Castel J,Denis RG,Rouch C,Delbes AS,Martinez S,et al.Dietary triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspects of feeding.Mol Psychiatry 2014;19:1095-105 Carlin J,Hill-Smith TE,Lucki I,Reyes TM.Reversal of dopamine system dysfunction in response to high-fat diet.Obesity(Silver Spring)2013;21:2513-21. De Wouters d’Oplinter A,Rastelli M,Van Hul M,Delzenne NM,Cani PD,Everard A.Gut microbes participate in food preference alterations during obesity.Gut Microbes 2021;13:1959242. Decarie-Spain L,Hryhorczuk C,Lau D,Jacob-Brassard E,Fisette A,Fulton S.Prolonged saturated,but not monounsaturated,high-fat feeding provokes anxiodepressive-like behaviors in female mice despite similar metabolic consequences.Brain Behav Immun Health 2021;16:100324. Delbes AS,Castel J,Denis RGP,Morel C,Quinones M,Everard A,et al.Prebiotics Supplementation Impact on the Reinforcing and Motivational Aspect of Feeding.Front Endocrinol(Lausanne)2018;9:273. Delzenne NM,Neyrinck AM,Backhed F,Cani PD.Targeting gut microbiota in obesity:effects of prebiotics and probiotics.Nature Reviews Endocrinology 2011;7:639-46. Depommier,C.,et al.,Pasteurized Akkermansia muciniphila increases whole-body energy expenditure and fecal energy excretion in diet-induced obese mice.Gut Microbes,2020.11(5):p.1231-1245.Depommier C,Everard A,Druart C,Plovier H,Van Hul M,Vieira-Silva S,et al.Supplementation with Akkermansia muciniphila in overweight and obese human volunteers:a proof-of-concept exploratory study.Nat Med 2019;25:1096-103. Erny,D.,et al.,Host microbiota constantly control maturation and function of microglia in the CNS.Nat Neurosci,2015.18(7):p.965-77. Everard A,Belzer C,Geurts L,Ouwerkerk JP,Druart C,Bindels LB,et al.Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.Proc Natl Acad Sci U S A 2013;110:9066-71. Everard A,Plovier H,Rastelli M,Van Hul M,de Wouters d’Oplinter A,Geurts L,et al.Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis.Nat Commun 2019;10:457. Frank G.,Altered Brain Reward Circuits in Eating Disorders:Chicken or Egg? Curr Psychiatry Rep 2013;15:396. Guillemot-Legris O,Muccioli GG.Obesity-Induced Neuroinflammation:Beyond the Hypothalamus.Trends Neurosci 2017;40:237-53. Karcher N,Nigro E,Puncochar M,Blanco-Miguez A,Ciciani M,Manghi P,Zolfo M,Cumbo F,Manara S,Golzato D,Cereseto A,Arumugam M,Bui TPN,Tytgat HLP,Valles-Colomer M,de Vos WM,Segata N.Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly.Genome Biol.2021 Jul 14;22(1):209. Plovier H,Everard A,Druart C,Depommier C,Van Hul M,Geurts L,et al.A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice.Nat Med 2017;23:107-13. Rogers P.J.Food and drug addictions:Similarities and differences.Pharmacology,Biochemistry and Behavior 2017;153:182-190. Rossi,C.,et al.,Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis.Cell Death Dis,2018.9(2):p.250. van de Wouw M,Schellekens H,Dinan TG,Cryan JF.Microbiota-Gut-Brain Axis:Modulator of Host Metabolism and Appetite.J Nutr 2017;147:727-45. Volkow ND,Wang GJ,Fowler JS,Telang F.Overlapping neuronal circuits in addiction and obesity:evidence of systems pathology.Philos Trans R Soc Lond B Biol Sci 2008;363:3191-200. Volkow ND,Wang GJ,Fowler JS,Tomasi D,Baler R.Food and Drug Reward:Overlapping circuits in human obesity and addiction.Curr Topics Behav Neurosci 2012;11:1-24. Wang GJ,Volkow ND,Logan J,Pappas NR,Wong CT,Zhu W,et al.Brain dopamine and obesity.Lancet 2001;357:354-7. Zhao J,Bi W,Xiao S,Lan X,Cheng X,Zhang J,et al.Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice.Sci Rep 2019;9:5790.

Claims

1. A composition comprising one or more bacteria of the genus Akkermansia and / or extracts thereof and / or fragments thereof for use in the prevention and / or treatment of reward system dysregulation.

2. 2. The composition for use according to claim 1, wherein the bacterium is Akkermansia muciniphila or Akkermansia species, and combinations thereof.

3. 2. The composition for use according to claim 1, wherein the reward system dysregulation is selected from the group comprising or consisting of psychiatric disorders, neurological disorders, disorders due to side effects of treatment, and combinations thereof.

4. 4. The composition for use according to claim 3, wherein the psychiatric disorder is selected from the group comprising or consisting of addiction-related disorders, eating-related disorders, affective disorders, obsessive-compulsive disorders, schizophrenia, attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, anxiety disorders, and the like.

5. 5. The composition for use according to claim 4, wherein the addiction-related disorder is selected from the group including or consisting of alcohol-related addiction, drug-related addiction, gaming-related addiction, and the like.

6. 4. The composition for use according to claim 3, wherein the neurological disorder is selected from the group comprising or consisting of Parkinson's disease, Tourette's syndrome, and the like.

7. 4. The composition for use according to claim 3, wherein the disorders due to side effects of treatment are selected from the group comprising or consisting of gaming addiction, shopping addiction, food addiction such as bulimia, hypersexuality, etc.

8. 5. The composition for use according to claim 4, wherein the eating disorder comprises or is selected from the group consisting of bulimia nervosa, binge eating disorder, anorexia nervosa (including restrictive and binge eating / purging types), pica, rumination disorder, purging disorder, night eating syndrome, avoidant-restrictive food intake disorder, overweight-related disorders and obesity-related disorders, food addiction, eating addiction, food craving, food seeking, compulsive eating disorder, impulsive eating disorder, failure of a calorie-restricted diet, weight loss non-responder or non-responder to dietary advice for weight loss, etc.

9. The composition for use according to claim 1 , wherein the composition further comprises one or more active agents.

10. 10. The composition for use according to claim 9, wherein the active agent is a therapeutic or nutritional agent.

11. 10. The composition for use according to claim 9, wherein the active agent is a beneficial microorganism selected from the group comprising or consisting of bacteria from the genera Verrucomicrobiaceae, Tannerellaceae, Clostridiaceae, Peptostreptococcusceae, Prevotellaceae, Methylobacteriaceae, Parabacteroides, Turicibacter, Coprococcus, Noelia, Prevotella, and Staphylococcus.

12. The composition for use according to any one of claims 1 to 11, wherein the composition is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

13. The composition for use according to any one of claims 1 to 11, wherein the composition is in the form of a nutritional composition further comprising a nutritionally acceptable carrier.

14. A composition comprising one or more bacteria of the genus Akkermansia and / or extracts thereof and / or fragments thereof for use as an adjuvant in a treatment administered to a subject suffering from a reward system dysregulation disorder.

15. The composition for use according to any one of claims 1 to 11 or 14, wherein the composition is contained in a kit, the kit further comprising a means for administering the composition.

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