Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K2035/11—Medicinal preparations comprising living procariotic cells
  • A61K2035/115—Probiotics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/02—Atmosphere, e.g. low oxygen conditions
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/32—Amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/74—Undefined extracts from fungi, e.g. yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/80—Undefined extracts from animals

Definitions

• the invention relates to the prevention and treatment of chronic inflammatory diseases, inflammatory gastrointestinal diseases and / or cancers with specific bacteria of the intestinal microbiota and compositions containing them.
• Inflammation is the body's normal defense process in the face of attack. It makes it possible to fight and eliminate the foreign agent (s) at the origin of the said aggression.
• This inflammation known as acute, is reversible and has only a limited duration ranging from a few minutes to a few days. However, in some cases, the inflammatory process goes wrong and the inflammation becomes chronic. Instead of contributing to the body's defense, the actors involved in the inflammatory process become dangerous and lead to an often serious and disabling pathological condition.
• Chronic inflammatory diseases are therefore diseases whose physiopathology is directly linked to inflammation, said inflammation being of autoimmune and / or auto-inflammatory origin.
• All organs can be affected by chronic inflammation, such as for example the digestive system (Crohn's disease, ulcerative colitis, autoimmune gastritis, etc.), the liver (hepatitis, NAFLD, non-alcoholic steatohepatitis, etc.). ), pancreas (pancreatitis), lungs (asthma), skin (atopic dermatitis such as psoriasis), nervous system (multiple sclerosis), joints (polyarthritis).
• chronic inflammation is also present in other situations where other mechanisms are involved, such as cancer in particular.
• Chronic inflammation and in particular chronic inflammatory diseases, affect a large portion of the population, in varying proportions depending on the pathology concerned.
• the consequences for health are major, because these diseases, although they develop slowly, are disabling, painful and associated with a high risk of early mortality.
• - purine metabolism modulators which are immunomodulators, such as methotrexate and azathioprine,
• - biological drugs such as anti-TNF- ⁇ , antagonists of IL1 and IL6 receptors, or even
• applications WO2018 / 162738 and WO2018 / 162726 teach that the use of particular bacteria of the Christensenellaceae family, belonging to species defined as being specifically distant from those of the Christensenella genus and in particular of the Christensenella minuta and Christensenella species timonensis, make it possible to fight in particular against overweight and obesity, by intervening for example on the presence of visceral fat and on intestinal permeability.
• Akkermansia muciniphila belonging to the Verrucomicrobiaceae family and to the Akkermansia genus, is a bacterium identified for the first time in 2004 and which represents approximately 1 to 3% by number of the total of the bacterial genera of the intestinal microbiota of healthy adults. health (Derrien et al., 2004, 54: 1469-1476; Derrien et al. Applied Environmental Microbiology 2008, 74, 1646-1648).
• Akkermansia muciniphila The population in number of Akkermansia muciniphila has already been reported to be reduced in stool samples from obese individuals and patients with inflammatory gastrointestinal disease (Png, et al. Gastroenterol. 2010; 105 : 2420-2428). Akkermansia muciniphila is associated with a healthier metabolic state and better clinical outcomes after calorie restriction intervention in overweight or even obese adults.
• bacteria of the genus Christensenella and in particular the species Christensenella minuta, Christensenella massiliensis and Christensenella timonensis, when they are administered to humans or animals, are capable of 'act on the markers of inflammation at the origin of inflammatory diseases, in particular inflammatory gastrointestinal diseases and chronic inflammatory diseases; and cancers.
• the inventors unexpectedly determined synergistic anti-inflammatory properties resulting from the use of a bacterium of the genus Christensenella in association with a bacterium of the genus Akkermansia, in particular Akkermansia muciniphila, a well-known new generation probiotic (NGP) from the Verrucomicrobiaceae family.
• NTP new generation probiotic
• the invention relates to a bacterium of the genus Christensenella, for its use in the prevention and / or treatment of chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers in human or animal.
• such a bacterium when it is administered to a human being or an animal exhibiting chronic inflammation, is capable of acting on molecules produced in excess during chronic inflammation such as kynurenine, interleukin 6, interleukin 8, TNFalpha, interleukin lb, lipocalins or fecal calprotectin, and / or by stimulating the production of the anti-inflammatory cytokine interleukin 10.
• a subject of the invention is also compositions comprising at least one bacterium of the genus Christensenella, alone or in combination with at least one additional bacterium, preferably at least one additional bacterium of the genus Akkermansia, for its use in prevention and / or the treatment of chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers in humans or animals.
• composition comprising, in a physiologically acceptable medium, at least one of these bacteria (at least one bacterium of the genus Christensenella alone or in combination with at least one additional bacterium of the genus Akkermansia) and / or their supernatant, is also referred to per se.
• Figure 1 shows the production of interleukin-8 (IL-8) (in pg / mL) (ordinate) in HT-29 cells stimulated by TNF-a in the presence of strains of Christensenella minuta, Christensenella timonensis , Akkermansia municiphila, Christensenella minuta + Akkermansia municiphila and Christensenella timonensis + Akkermansia municiphila (abscissa).
• IL-8 interleukin-8
• FIG. 2 represents the secretion of IL-8 by the intestinal cells of the HT29 line after co-incubation in the presence of supernatants of strains of Christensenella minuta ('DSMZ: DSM22607) and stimulation by TNF-a.
• Dunn's nonparametric statistical test * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
• FIG. 3 represents the measurement of the transepithelial resistance on intestinal cells of the Caco-2 line after co-incubation in the presence of Christensenella minuta bacteria (DSMZ: DSM22607) and after stimulation with TNF- a.
• DSMZ Christensenella minuta bacteria
• TNF- a Tumor Necrosis Factor alpha. Means of 3 experiments repeated in triplicate.
• Figure 4 shows the effects of Christensenella minuta (DSM 22607) on an inflammation model induced by DNBS.
• DSM 22607 Monitoring of the weight loss of the treated groups from the day of the injection (D0).
• B Significant decrease in macroscopic scores indicating a reduction in the severity of inflammation following DSM 22607 and 5ASA treatments.
• C myeloperoxidase activity
• IL-10 interleukin-10
• D Nonparametric ANOVA followed by Dunn's statistical test: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
• DNBS Dinitrobenzene sulfonic acid.
• Figure 5 demonstrates the generalization of the anti-inflammatory effects of the species of Christensenella minuta on a model of inflammation induced by DNBS.
• A Reduction in macroscopic scores indicating a reduction in the severity of inflammation following treatments with different species of Christensenella minuta.
• B Immunomodulatory effects of different species of Christensenella minuta through the decrease in myeloperoxidase activity (C). Nonparametric ANOVA followed by Dunn's statistical test: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
• DNBS Dinitrobenzene sulfonic acid.
• FIG. 6 represents the effect of the supernatants of the strains of Christensenella minuta (DSMZ: DSM22607, C. minuta 1 and C. minuta 2) on the proliferation of HT-29 cells (adenocarcinoma of the colon).
• RLU Relative Luminescence Unit.
• bacteria also means the term “bacterial strains”.
• a bacterial strain being to be understood as being a bacterium of a given strain belonging to a particular species, for example the species Christensenella minuta. Mention may be made, for example, of the bacterial strain or bacterium Christensenella minuta DSM22607.
• the two terms can be used interchangeably to designate a bacterium (s) in the context of the invention.
• hypoproduction of interleukin 6 within the meaning of the invention, is meant an excessive production of interleukin 6 relative to the production in a healthy person or animal without inflammation.
• hypoproduction of interleukin 8 within the meaning of the invention, is meant an excessive production of interleukin 8 relative to the production in a healthy person or animal without inflammation.
• under-production of interleukin 10 within the meaning of the invention, is meant an insufficient production of interleukin 10 relative to the production in a healthy person or animal without inflammation.
• chronic inflammatory disease within the meaning of the invention, is meant in particular any chronic disease whose pathophysiology, slow and progressive, is directly related to inflammation of autoimmune and / or auto. inflammatory.
• inflammatory gastrointestinal disease within the meaning of the invention is understood to mean in particular an inflammatory bowel disease, more particularly an inflammatory bowel disease of the colon.
• Said colonic inflammatory bowel disease can, in particular, be selected from the group consisting of Crohn's disease (CD), ulcerative colitis (UC) and pouchitis, in particular CD and UC.
• CD and UC are two diseases characterized by inflammation of the lining of part of the digestive tract, linked to an overactive digestive immune system.
• prevent or “prevention” within the meaning of the invention, is meant the reduction to a lesser degree of the risk or the probability of occurrence of a given phenomenon, that is to say, in in the context of the present invention, chronic inflammation, gastrointestinal inflammation and pathologies arising therefrom such as cancer, in particular intestinal inflammation.
• treating or “treatment” within the meaning of the invention, is meant a decrease in the progression of the disease, a stabilization, a reversal or regression, or even an interruption or inhibition of the progression of an inflammatory disease. chronic, inflammatory gastrointestinal disease, cancer. In the context of the invention, these The terms also apply to one or more symptoms of said diseases of the present invention.
• non-deficient in Christensenella in the intestinal microbiota, within the meaning of the present invention, is meant a human being or an animal whose bacteria of the genus Christensenella in his intestinal microbiota represents at least 0.01% by number. of the total of the bacterial genera detected in its intestinal microbiota, in particular at least 0.1%, and preferably at least 0.5%. Collected in the stool, the abundance of Christensenella can for example be measured by a quantitative sequencing assay, fluorescent in situ hybridization (FISH), qPCR (quantitative PCR) or by metagenome analyzes (relative quantification) , well known to those skilled in the art.
• FISH fluorescent in situ hybridization
• qPCR quantitative PCR
• metagenome analyzes quantitative quantification
• physiologically acceptable medium within the meaning of the invention is meant a medium which is compatible with the organism of the individual to which said composition is to be administered. It can be, for example, a non-toxic solvent such as water. In particular, said medium is compatible with oral administration.
• microbiota is meant all the microorganisms that have colonized an individual and with which he cohabits: bacteria for the most part, but also viruses, fungi, yeasts and protozoa.
• the composition of the microbiota differs according to the colonized surfaces: we thus distinguish the cutaneous microbiota, the vaginal microbiota, the urinary microbiota, the respiratory microbiota, the ENT microbiota (otorhinolaryngology) and the intestinal microbiota, formerly called the intestinal flora, by far the most important with its 100,000 billion germs.
• intestinal microbiota is meant all the microorganisms, in particular bacteria, which inhabit the intestine of a given individual.
• body mass index is meant, according to an official definition of the World Health Organization (WHO), the indicator of the health risks associated with excessive weight and weight. insufficient. BMI is calculated by dividing an individual's weight (in kilograms) by their height (in meters) squared. A BMI value is associated with a specific body size according to the classification given by the WHO (https://www.who.int/features/factfiles/obesitv/facts/en/).
• an effective amount refers to an amount of an active ingredient which is effective to relieve or to some extent reduce one or more of the symptoms of the disease in the need for treatment, or to delay the onset of clinical markers or symptoms of a disease which need prevention, when the compound is administered.
• the amount refers to an amount of the active ingredient which exhibits effects such as (i) reversing the rate of progression of a disease, (ii) inhibiting the progression of the disease to some extent, and / or, (iii) relieve to some extent (or, preferably, by eliminating) one or more symptoms associated with the disease.
• the effectiveness of the amount can be determined empirically by testing the relevant compounds in known in vivo and in vitro model systems for a disease requiring treatment.
• the subject of the invention is therefore the use in the prevention and / or treatment of chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers, in particular cancers of an inflammatory nature, in 'human being or animal, of at least one bacterium of the Christensenellaceae family, preferably of the genus Christensenella or of a composition comprising at least one such bacterium and / or a culture supernatant of at least said bacterium of the genus Christensenella.
• the bacteria can be any bacterial strain of one of the species of the genus Christensenella.
• said composition further comprising an additional bacterium of the Verrucomicrobiaceae family and / or a culture supernatant of said additional bacterium, in particular said bacterium is of the Akkermansia genus, more particularly of the Akkermansia muciniphila species.
• the invention therefore relates to a bacterium of the Christensenellaceae family, in particular of the genus Christensenella for its use in the prevention and / or treatment of inflammatory diseases, preferably of chronic inflammatory diseases and / or of inflammatory gastrointestinal diseases. and / or cancer in humans or animals, in particular in people or animals with an inflammatory disease or cancer with a hyperproduction of interleukin 6 and / or a hyperproduction of interleukin 8 and / or a underproduction of interleukin 10.
• bacteria of the genus Christensenella when administered to a human being or an animal exhibiting chronic inflammation, gastrointestinal inflammation or cancer, are capable of acting on the molecules produced in excess in chronic inflammation, gastrointestinal inflammation or cancer, in particular on interleukin 6, interleukin 8 and kynurenine, but also on the TNFalpha, interleukin lb, lipocalins or fecal calprotectin.
• the inventors have discovered that bacteria of the genus Christensenella have the unexpected ability to reduce in vitro and in vivo inflammation and cell proliferation, in particular gastrointestinal inflammation and more particularly chronic inflammation of the intestine.
• the bacteria according to the present invention are capable of significantly reducing the production of pro-inflammatory molecules, such as interleukin 6 (IL-6) and interleukin 8 (IL-8).
• the bacteria or bacteria useful according to the invention are administered to humans or animals in an amount effective for an action on at least one of these markers of inflammation, that is that is to say to decrease the production of at least one of these molecules in the body.
• the bacteria or bacteria can be administered at a rate of 10 7 to 10 11 colony forming units (CFU) per day, regardless of the weight of the person or the animal, preferably a dose of 10 9 to 10 12 CFU per day is administered, more preferably a dose equal to 10 9 CFU.
• the dose comprises at least one bacterium chosen from Christensenella timonensis, Christensenella minuta, Christensenella massiliensis and / or their mixtures.
• it is a single dose, that is to say administered all at once or a dose before each meal, ie three times a day.
• the bacterium of the Christensenellaceae family in particular of the genus Christensenella is useful in the treatment of at least one chronic inflammatory disease chosen from chronic inflammatory diseases of the intestine, chronic inflammatory diseases of the liver, chronic inflammatory diseases of the pancreas, polyarthritis, atopic dermatitis, neuro-inflammatory diseases, chronic obstructive pulmonary disease.
• it has an effect in the prevention and / or treatment of at least one disease selected from Crohn's disease, ulcerative colitis, pouchitis, ulcerative colitis, celiac disease, autoimmune gastritis, hepatitis, nonalcoholic steatohepatitis, primary sclerosing cholangitis, pancreatitis, rheumatoid arthritis, psoriatic arthritis, psoriasis, l 'eczema.
• at least one disease selected from Crohn's disease, ulcerative colitis, pouchitis, ulcerative colitis, celiac disease, autoimmune gastritis, hepatitis, nonalcoholic steatohepatitis, primary sclerosing cholangitis, pancreatitis, rheumatoid arthritis, psoriatic arthritis, psoriasis, l 'eczema.
• the bacterium of the Christensenellaceae family in particular of the genus Christensenella, is useful in the treatment of at least one inflammatory gastrointestinal disease such as an inflammatory bowel disease, more particularly an inflammatory bowel disease of the colon.
• inflammatory bowel disease such as an inflammatory bowel disease, more particularly an inflammatory bowel disease of the colon.
• the term “disease” is understood to mean a disease or a pathology.
• Said colonic inflammatory bowel disease can in particular be selected from the group consisting of Crohn's disease, ulcerative colitis and pouchitis, in particular from the group consisting of Crohn's disease and ulcerative colitis. hemorrhagic.
• the bacterium of the Christensenellaceae family in particular of the Christensenella genus is useful in the treatment of a proliferative disease chosen from lymphomas, glioblastomas, myelomas, leukemias, colorectal cancers, cancers of the breast, prostate, ovary, uterus, pancreas, lungs, liver, gallbladder, and kidneys.
• a proliferative disease chosen from lymphomas, glioblastomas, myelomas, leukemias, colorectal cancers, cancers of the breast, prostate, ovary, uterus, pancreas, lungs, liver, gallbladder, and kidneys.
• the bacteria or bacteria useful according to the invention are bacteria of the genus Christensenella. It may in particular be Christensenella massiliensis, Christensenella timonensis, Christensenella intestinihominis, and / or Christensenella minuta. According to a particularly suitable variant, it is Christensenella minuta.
• the bacteria or bacteria useful according to the invention are preferably administered in a composition.
• Said composition comprising, in a physiologically acceptable medium, at least the useful bacterium (s) and / or their supernatant.
• the invention also relates to a composition
• a composition comprising at least one bacterium of the genus Christensenella in the prevention and / or treatment of chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers, in humans or animals, preferably in people or animals with hyperproduction of interleukin 6.
• the invention makes it possible to provide an effective amount of bacteria of the Christensenellaceae family, such as Christensenella massiliensis, Christensenella timonensis, Christensenella intestinihominis and / or Christensenella minuta, for use in the treatment:
• - diseases associated with increased interleukin 6 such as inflammatory disorder, allergic disorder, autoimmune disorder, transplant rejection disorder, atherosclerosis, osteoporosis, neuropathic pain, sepsis , Castleman's disease, a fibrotic condition, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis or ankylosing spondylitis,
• interleukin 8 diseases associated with an increase in interleukin 8 such as atopic dermatitis, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, stroke, septic shock, endotoxic shock, psoriasis, Gram-negative sepsis, toxic shock syndrome, renal reperfusion injury, glomerulonephritis, thrombosis, graft versus host reaction, allograft rejection, malaria, restenosis, angiogenesis, atherosclerosis, osteoporosis, gingivitis and unwanted release of hematopoietic stem cells, diseases caused by respiratory viruses, herpes virus and hepatitis viruses, meningitis, herpetic encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoid hemorrhage, post-surgical trauma, cystic fibrosis, pruritus, interstitial pneumonia, hypersensitivity, crystalline arthritis, Lyme
• interleukin 10 diseases associated with a decrease in interleukin 10 or which can be treated by increasing interleukin 10 such as autoimmune diseases, rejection of organ and bone marrow transplants, graft versus host disease , parasitic infections, granulomas, Crohn's disease, colitis, pancreatitis, inflammatory lung disease, inflammatory eye disease, atopic dermatitis and rhinitis.
• the invention relates to a composition for its use in the treatment and / or prevention of chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers in a human being or a patient. animal, said composition comprising, in a physiologically acceptable medium (i) at least bacteria of the genus Christensenella, and / or (ii) a culture supernatant of at least one bacterium of the genus Christensenella.
• the present invention also relates to a composition for its use in the treatment and / or prevention of an inflammatory gastrointestinal disease in an individual, said composition comprising, in a physiologically acceptable medium
• At least one bacterial strain chosen from the group consisting of Christensenella timonensis, Christensenella minuta and / or mixtures thereof,
• the present composition can be administered to a human being suffering from chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers, said human being having an index of body mass (BMI) less than 25, in particular said composition is administered to a human being suffering from inflammatory gastrointestinal diseases.
• BMI less than 25 denotes a BMI strictly less than 25, more particularly a BMI less than or equal to 24.9.
• said human being may in particular have a BMI of less than 25 and greater than or equal to 18.5.
• said composition according to the invention is characterized in that the bacterium of the genus Christensenella is chosen from Christensenella massiliensis, Christensenella timonensis and / or Christensenella minuta and / or Christensenella intestinihominis and / or their mixtures.
• the composition can be used in an individual who is not deficient in Christensenella in the intestinal microbiota, in particular in a human being or an animal whose genus Christensenella represents at least 0.01. % in number of the total of the bacterial genera of its intestinal microbiota.
• the bacteria or bacteria are present in an effective amount in the composition allowing an effect on chronic inflammatory diseases and / or inflammatory gastrointestinal diseases and / or cancers from which the persons or animals treated are affected.
• person is meant an individual, in particular a human being or an animal.
• the composition useful according to any one of the preceding embodiments comprises 10 6 to 10 12 colony-forming units (CFU) of bacteria of the genus Christensenella per daily dose to be administered of said composition.
• CFU colony-forming units
• this corresponds to a daily dose of bacteria to be administered, whatever the weight of the person or the animal.
• this dose is administered all at once.
• the useful composition comprises 10 7 to 10 11 CFU of bacteria from genus Christensenella per daily dose to be administered, preferably 10 9 CFU.
• said bacteria present in the composition may be of the same species, of a mixture of species or of a mixture of bacterial strains.
• the composition useful according to the invention can be in liquid form.
• bacteria of the Christensenellaceae family in particular of the Christensenella genus and / or a culture medium for said bacteria which makes it possible to preserve them, such as, for example, preferably the anaerobic Columbia agar medium enriched with sheep blood, or a medium equivalent that does not contain any derivative of animal origin.
• culture medium means the culture supernatant in which the bacteria or the bacterial strains have resided during their culture, or extracellular medium.
• the composition useful according to the invention may be in solid form.
• the bacteria can be present in lyophilized form, and can also include excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum , calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, sorbitol and / or glycerol.
• excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum , calcium lactate, magnesium
• compositions useful according to the invention may be in the form of powder, microencapsulated powder, gelatin capsule, capsule, tablet, lozenge, granules, emulsion, suspension, suppository, a food product, beverage, pharmaceutical, nutraceutical, food additive, dietary supplement or dairy product.
• they may be in a gastro-resistant form, such as a coated tablet containing microencapsulated bacteria.
• Said composition can thus be provided with a coating resistant to gastric juice, in order to ensure that the bacteria of the invention included in said composition can pass through the damaged stomach. The release of the bacteria (s) can thus occur for the first time in the upper intestinal tract.
• composition according to the invention can be included in a food supplement.
• a food supplement for administration by the oral may be present in capsules, hard capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes, lozenges, gums, drinkable solutions or emulsions, syrup or gel.
• a food supplement according to the present invention can further comprise a sweetener, a stabilizer, an antioxidant, an additive, a flavoring agent and / or a colorant.
• the formulation thereof is carried out by means of the usual processes for producing sugar-coated tablets, hard capsules, gels, controlled release hydrogels, emulsions, tablets or capsules.
• a composition according to the present invention can also be in the form of a nutritional composition.
• a nutritional composition according to the present invention is in the form of a yogurt, a cereal bar, a breakfast cereal, a dessert, a frozen food, a soup, a pet food, a liquid suspension. , a powder, a tablet, a gum or a candy.
• a composition according to the present invention can further comprise at least one ingredient chosen from: antioxidants, fish oils, DHA, EPA, vitamins, minerals, phytonutrients, a protein, a lipid, probiotics and combinations of these.
• the bacteria according to the invention alone or included in the composition according to any one of the preceding embodiments can be used alive, semi-active, inactivated, or dead, preferably in a living form. Bacteria in semi-active, inactivated or dead form can be, for example, by irradiation, thermal inactivation or lyophilization, in particular by heat, exposure to an appropriate pH, UV, gamma rays, X rays or to high pressure.
• the term “semi-active” thus designates a bacterium with low physiological activity, the capacity of which to proliferate is reduced, temporarily or permanently.
• the term “inactivated” designates a bacterium which is no longer able, temporarily or permanently, to proliferate.
• the term “dead” designates a bacterium which is no longer able, definitively, to proliferate. Dead or inactivated bacteria can have cell membranes intact or ruptured. Thus the term “inactivated” also designates the extracts and lysates of bacteria obtained.
• a bacterium according to the invention alone or in the composition according to the invention can be used in whole form, that is to say essentially in its native form, or in the form of extracts or lysates comprising fractions and / or metabolites of this microorganism.
• a lysate can denote the whole of the lysate obtained by lysis or only a fraction of the latter and can be prepared according to one of the conventional methods well known to those skilled in the art such as for example a thermal shock, by ultrasound, a osmotic shock, or under mechanical stress, such as by centrifugation. They can be all alive, all semi-active, all inactivated or all dead. In particular, it can be a mixture of living, semi-active, inactivated and dead bacteria.
• At least some of the bacteria are alive, in particular at least 50% (by number), even more preferably at least 90% (by number).
• the bacteria present in the composition useful according to the invention are for at least 50% living bacteria (in number), preferably for at least 90% of living bacteria (in number) , even more preferably all alive.
• the storage conditions according to the invention are provided for liquid formulations in the form of a frozen product maintained at -20 ° C in a sealed bag.
• the storage conditions according to the invention comprise a capsule or a coating hermetic to light and to oxygen maintained at an ambient temperature of between 15 ° C and 40 ° C and a humidity of between 3% and 70%.
• a composition according to any one of the preceding embodiments is provided for the digestive tract, in particular the intestine. Consequently, the bacteria useful according to the invention, and in particular the compositions including it, can be administered orally, topically, inhaled or rectally, preferably rectally or intrarectally.
• rectal or intrarectal administration is carried out in the form of a suppository, an enema or a foam.
• the inventors have also determined, surprisingly, the ability of strains of Christensenella to increase the anti-inflammatory properties of a strain of Akkermonsio, and in particular of the strain Akkermansia muciniphila.
• the inventors have demonstrated the existence of a synergistic effect of a combination of a strain of Christensenella (or of a culture supernatant of such a strain) and of a strain of Akkermansia (or of a culture supernatant of such a strain) on the prevention and / or treatment of gastrointestinal inflammation, and in particular the synergistic effect of a combination of a strain of Christensenella minuta and / or Christensenella timonensis (or culture supernatants of such strains) and a strain of Akkermansia muciniphila (or a supernatant thereof) on the prevention and / or treatment of gastrointestinal inflammation.
• composition can further comprise:
• the additional bacterium of the Verrucomicrobiaceae family is a bacterium of the genus Akkermansia, more particularly of the species Akkermansia muciniphila.
• bacterium of the species “Akkermansia muciniphila” is meant within the meaning of the invention also the bacterium called “Akkermansia municiphila”.
• the bacterium and the additional bacterium as mentioned above are, independently of one another, in a living, semi-active, inactivated and / or dead form, and are in particular in a living form.
• a composition according to the invention is suitable for the administration of a daily dose representing from 10 7 to 10 11 colony forming units (CFU), preferably a daily dose equivalent to 10 9 UFC, of a bacterium of the Verrucomicrobiaceae family, in particular of the Akkermansia genus, more particularly of an Akkermansia muciniphila bacterium.
• CFU colony forming units
• a composition according to the invention can in particular comprise a content of between 0.1 and 99% by weight, in particular from 10 to 90% by weight, in particular from 25 to 70% by weight and more particularly from 30 to 50% by weight, relative to the total weight of the composition.
• a composition according to any one of the aforementioned embodiments can comprise a content of between 0.1 and 99% by weight, in particular 10 to 90%, in particular 25 to 70% by weight, more particularly from 30 to 50% by weight of supernatant relative to the total weight of the composition.
• compositions useful according to the invention in addition to the bacteria useful according to the invention, can comprise at least one probiotic, and / or at least one prebiotic.
• compositions useful according to the invention in addition to the bacteria useful according to the invention can comprise other compounds, such as: - at least one probiotic, and / or
• At least one bacterium producing lactic acid which makes it possible to create an anaerobic environment favorable to Christensenellaceae, such as at least one bacterium chosen from bacteria of the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and / or at least one other organism promoting the anaerobic conditions necessary for the survival of Christensenellaceae, such as at least one yeast chosen from Saccharomyces spp. or microorganisms of the Methanobacteriaceae family and / or
• At least one bacterium associated with the ecosystem of Christensenellaceae because they facilitate their survival in the intestine such as at least one bacterium chosen from bacteria of the phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, and Verrucomicrobia, and / or
• At least one bacterium chosen from bacteria of the order Clostridales, Verrucomicrobiales, Aeromonadales, Alteromonadales, ML615J-28, RF32, YS2, from the Clostridiaceae family, Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, Enterococcaceae , Rikenéllaceae, Dehalobacteriaceae, Veillonellaceae and / or
• Faecalibacterium chosen from bacteria of the genus Faecalibacterium, Akkermansia, Eubacterium and Oscillospira such as for example Faecalibacterium prausnitzii, Akkermansia muciniphila, Eubacterium halii, Oscillospira guilliermondii., and / or
• At least one prebiotic such as for example at least one prebiotic chosen from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and / or beta-caseins, and / or
• At least one polyphenol such as for example at least one polyphenol chosen from quercetin, kaempferol, resveratrol, flavones (such as luteolin), flavan-3-ols (such as catechins), flavanones (such as narinenin) ), isoflavones, anthocyanidins, proanthocyanidins, and / or
• non-steroidal anti-inflammatory drugs antibodies directed against pro-inflammatory targets (anti-TNFalpha), antirheumatic drugs, analgesics, antimicrobials, corticosteroids, anabolic steroids, antidiabetics, thyroid agents, antidiarrhoeals, cough suppressants, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietin, immunoglobulins, immunosuppressants, growth hormones, hormonal drugs, estrogen receptor modulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, anti-depressants, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma drugs, beta-agonists, inhaled steroids, leukotriene inhibitors, cromoglycates or cromoglycidic acids, epinephrine, alpha dornase
• anti-TNFalpha pro-inflammatory targets
• the present invention relates to a composition
• a composition comprising, in a physiologically acceptable medium:
• (I) (a) at least one bacterium selected from the group consisting of Christensenella timonensis, Christensenella minuta, Christensenella massiliensis, Christensenella intestinihominis, and / or mixtures thereof;
• the present invention relates to a method for reducing and / or preventing inflammation in an individual in need thereof, comprising the administration of an effective amount of a bacterium of the Christensenellaceae family to said individual, in particular a bacterium of the species Christensenella.
• the invention also relates to a method for reducing the level of IL-6 and / or stabilizing the level of IL-6 and / or preventing an increase in the level of IL-6 in an individual in need thereof, comprising administering an effective amount of a bacterium of the Christensenellaceae family to said individual, in particular a bacterium of the species Christensenella.
• said individual exhibiting high serum levels of IL-6 before treatment.
• said elevated serum IL-6 levels are greater than 5 pg / ml, greater than 10 pg / ml, greater than 20 pg / ml, greater than 30 pg / ml, greater than 40 pg / ml, greater than 50 pg / ml, greater than 70 pg / ml, greater than 90 pg / ml or greater than 100 pg / ml.
• the invention also relates to a method for reducing the level of IL-8 and / or stabilizing the level of IL-8 and / or preventing an increase in the level of IL-8 in an individual in need thereof, comprising administering an effective amount of a bacterium of the Christensenellaceae family to said individual, in particular a bacterium of the species
• said individual exhibiting high serum IL-8 levels before the treatment.
• said elevated serum IL-8 levels are greater than 5 pg / ml, greater than 10 pg / ml, greater than 20 pg / ml, greater than 30 pg / ml, greater than 40 pg / ml, greater than 50 pg / ml, greater than 70 pg / ml, greater than 90 pg / ml or greater than 100 pg / ml.
• the invention also relates to a method for increasing the level of IL-10 and / or stabilizing the level of IL-10 and / or preventing a decrease in the level of IL-10 in an individual in need thereof, comprising administering an effective amount of a bacterium of the Christensenellaceae family to said individual, in particular a bacterium of the species
• said individual exhibiting low serum IL-10 levels before treatment.
• said low serum IL-10 levels are less than 15 pg / ml, less than 10 pg / ml or less than 5 pg / ml.
• the invention relates to a method for reducing the level of kynurenine and / or stabilizing the level of kynurenine and / or preventing an increase in the level of kynurenine in an individual in need thereof, comprising the administration of a effective amount of a bacterium of the Christensenellaceae family to said individual, in particular a bacterium of the Christensenella species.
• said individual exhibiting high levels of fecal kynurenin before the treatment.
• the invention also relates to a method of treating and / or preventing cancer associated with inflammation in an individual in need thereof, comprising administering an effective amount of the Christensenellaceae bacteria to said individual.
• the method according to the present invention makes it possible to reduce the level of at least one of the markers of inflammation mentioned above in said individual, said marker being preferably reduced in serum, blood or stool.
• the present methods are implemented in an individual suffering from one or more diseases described above in the description. Said individual preferably being a human being.
• the Christensenella bacterium comprises a 16S ribosomal RNA gene exhibiting a sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or, preferably at least 90% with a 16S ribosomal RNA gene from Christensenella minuta, Christensenella massiliensis, or Christensenella timonensis, more preferably at least 97% sequence identity with a sequence selected from SEQ ID NO : 1, 2, B, 4, 5 or 6.
• SEQ ID NO: 1 is a sequence of the 16S ribosomal RNA gene of the bacterium Christensenella minuta DSM 22607, which has been isolated from a human fecal sample.
• the C. minuta 16S rRNA sequence is identified by the Genbank accession number AB490809.
• SEQ ID NO: 2 is a part of the 16S ribosomal RNA gene sequence from the bacterium Christensenella minuta DSM 22607. Additional 16S sequences for bacteria of the genus Christensenella are provided such as SEQ ID NOS: 3-6 (OTU numbers 701845, 177179, 1146771, and 361793 in the Greengenes database, respectively).
• each sequence with respect to SEQ ID NO: 1 is as follows: SEQ ID NO: 3, 93% identity with SEQ ID NO: 1; SEQ ID NO: 4, 95% identity with SEQ ID NO: 1; SEQ ID NO: 5, 97% identity with SEQ ID NO: 1; SEQ ID NO: 6, 95% identity with SEQ ID NO: 1.
• said methods comprise the administration of at least one bacterium or composition according to the present invention characterized by any one of the any embodiments described above, such as mixing or not the bacteria, the amount administered, the amount of viable bacteria, the additional components, for example active pharmaceutical ingredients, prebiotics or probiotics, vitamins, minerals, nutritional agents, other microorganisms, the particular formulation of said composition, for example powder, capsule, etc.
• the present methods include oral, inhaled, topical, rectal or intrarectal administration in a sufficient amount.
• Example 2 Christensenella massiliensis.
• Christensenella massiliensis can be cultivated according to the operating protocol described below.
• Christensenella timonensis can be cultivated according to the same operating protocol as that described in Example 2 for Christensenella massiliensis.
• Example 4 Specific strains according to the invention
• Example 5 Useful composition based on supernatants
• composition useful according to the invention is a composition comprising between 0.1 and 95% of culture supernatant of the strains Christensenella minuta DSM 22607, Christensenella timonensis DSM 102800 and Akkermansia municiphila DSM 22959.
• Example 6 Useful composition according to the invention in liquid form
• composition useful according to the invention in liquid form is a composition comprising Christensenella minuta 10 9 CFU / ml in the anaerobic RCM culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. .
• Example 6 The composition of Example 6 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella minuta 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
• RCB search cell bank
• the frozen composition must be warmed to room temperature until it returns to liquid form before use.
• Example 7 Useful composition according to the invention in solid form
• composition useful according to the invention in lyophilized form can be obtained by lyophilization of the composition of Example 6 in the frozen state.
• kynurenine The objective of this study is to demonstrate the anti-inflammatory effect in vitro of christensenella bacteria according to the invention. The demonstration of this effect was carried out on kynurenine, a known marker of inflammation. Human faeces were collected and cultured in a fermenter. The abundance of Christensenella spp. was correlated with the abundance of kynurenine present in the fermenter.
• - Faeces samples were diluted 1/5 (w / v) in phosphate buffered saline (IM) (PBS), pH 7.4.
• IM phosphate buffered saline
• PBS phosphate buffered saline
• the basic nutrient medium was prepared from 2g / L of soybean tryptone broth, 2g / L of yeast extract, 0.1g / L of NaCl, 0.04g / L of K2HPO4 , 0.01g / L of MgS0 3 .7H 2 0, 0.01g / L of CaCl 2 .6H 2 0, 2g / L NaHCOs, 0.5g / L of L-cystine HCl, 2m L / L of tween 80, 10pL / L of vitamin Kl, 0.05g / L of heme, 0.05g / L of bile salts, 4ml / L of resazarin (pH7)
• the 20mL biofermenters contained 18mL of basic nutrient medium autoclaved (121 ° C for 15 minutes) and poured aseptically into sterile biofermenters. This system was left to stand overnight with oxygen-free nitrogen bubbling through the medium at a rate of 2 mL / min.
• the pH was maintained between 6.7 and 6.9 using HCl or NaOH (0.5M).
• the temperature of each biofermenter was controlled at 37 ° C and the contents of the vessel homogenized with a magnetic mixer
• predigested proteins (0.35g) was added to the containers before inoculation with 2mL of fecal inocula at T0.
• the predigested proteins were obtained according to the gastrointestinal digestion protocol adapted from that of Versantvoort et al (2005). - the samples were collected before fermentation (T0) and after 48 hours of fermentation (T48), and frozen at -80 ° C until analyzes.
• the mixture was mixed and filtered through a 5-kDa cutoff filter to remove macromolecules.
• the metabolites were detected by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) analyzes.
• Relative peak area (peak area of a metabolite) / (peak area of internal standard x amount of sample).
• the DNA contained in the samples was extracted using the NucleoSpin ® 96 Soil kit from Macherey-Nagel according to the manufacturer's instructions.
• the library was then sequenced using 2 x 150 bp paired-end sequencing on an Illumina HiSeq platform.
• the objective of this study is to demonstrate the anti-inflammatory effect in vitro of bacteria according to Example 4.
• the demonstration was carried out on one of the markers of inflammation: interleukin 8 (IL-8 ).
• HT-29 human colon epithelial cells (LGC-Standars) were grown in Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) (Sigma-Aldrich) supplemented with 10% (w / v) heat-activated fetal bovine serum (FBS) (GibcoBRL, Eragny, France) and penicillin G / streptomycin (5000 lU / mL, 5000 pg / ml) (Sigma-Aldrich). Cell cultures were incubated in 25 cm 2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37 in a 5% (v / v) CO2 atmosphere until confluence.
• DMEM Dulbecco's Modified Eagle's Minimum Essential Medium
• FBS heat-activated fetal bovine serum
• penicillin G / streptomycin 5000 lU / mL, 5000 pg / ml
• 50,000 HeLa or HT-29 cells per well were seeded in 24-well culture plates (Nunc). Twenty-four hours before the bacterial contact, the culture medium was replaced with a medium containing 5% FBS. The experiments were conducted on day 7 after seeding, while the cells were confluent (l 83xl0 6 cells / well). Twenty-four hours before the bacterial co-culture (day 6), the culture medium was replaced with a medium containing 5% heat inactivated FBS and 1% glutamine. On the day of co-culture, 10% bacterial supernatant was added to DMEM in a total volume of 500 ⁇ l. The cells were stimulated
• Figure 1 represents the production of IL-8 in HT-29 cells stimulated by TNF -a in the presence of Christensenella minuta, Christensenella timonensis, Akkermansia muciniphila, Christensenella minuta + Akkermansia muciniphila and CChristensenella timonensis + Akkermansia muciniphila + Akkermansia muciniphila + Akkermansia muciniphila + Akkermansia muciniphila.
• the results are expressed in IL-8 (pg / mL) and were normalized using as reference value IL-8 produced after co-incubation with PBS as negative control. Results were analyzed using a Kruskal Wallis test followed by Dunns multiple comparison test (* p ⁇ 0.05; ** p ⁇ 0.01).
• the reference strain Akkermansia muciniphila DSM 22959 is well known for its immunomodulatory properties and more particularly for its anti-inflammatory effects (Derrien M, Belzer C, de Vos WM. Microb Pathog. 2017 May; 106: 171-181. doi: 10.1016 / j. micpath.2016.02.005. Epub 2016 Feb 11). To determine whether the strains of C.
• Christensenella minuta DSM 22607 and Christensenella timonensis DSM 102800 are capable of modulating the immune response, the inventors tested in vitro the immunomodulatory properties of the supernatants of the strains in a model based on the ability to block the production of IL -8 (a pro-inflammatory cytokine) induced by TNF- ⁇ stimulation in colon epithelial cells HT-29.
• IL -8 a pro-inflammatory cytokine
• Christensenella minuta DSM 22607 and Christensenella timonensis DSM 102800 increase the anti-inflammatory properties of Akkermansia muciniphila DSMZ 22959 in vitro.
• the objective of this study is to demonstrate the anti-inflammatory effect in vitro of bacteria according to the invention.
• the demonstration was carried out on one of the markers of inflammation: interleukin-8.
• DMEM Glutamax + 10% FCS is then removed to be replaced by medium with 5% FCS (JO).
• D + 1 the middle is removed.
• Two media DMEM Glutamax + 5% FCS +/- TNF-a are prepared. 450 ⁇ L / well of one of the two media are then added and then 50 ⁇ L of the supernatants to be tested. After 6 hours of co-incubation, the supernatants are collected and stored at -80 ° C.
• ELISA assays of the chemokine IL-8 are then carried out (ELISA MAX Deluxe set Human IL-8 from Biolegend).
• FIG. 2 represents the assay of the IL-8 chemokine secreted by HT-29 cells (colon adenocarcinomas) in an inflammatory state induced by TNF- ⁇ .
• Different bacterial supernatants C. minuta 1, C minuta 2, C. minuta B, C. minuta 4 and DSMZ, corresponding to the strain DSM22607 belonging to bacteria of the species Christensenella minuta were tested in order to evaluate their capacity immunomodulatory.
• the bacterial culture medium (GAM) represents the control making it possible to assess the effects of the supernatants. The experiment was carried out four times in triplicate.
• FIG. 3 represents the measurement of the trans-epithelial electrical resistance taken on Caco-2 epithelial cells. The latter by differentiating establish junctions between them (called tight junctions) to maintain the integrity of this barrier created by the cells, mimicking the intestinal barrier present in our digestive tract.
• cytokine TNF -a Tumor Necrosis Factor
• DMEM + TNF control group - at The "DMEM -" control group represents the wells where only the cell culture medium was present, without TNF -a or bacteria.
• Trans-epithelial electrical resistance is a method for quantifying the integrity of the barrier of epithelial tissue by measuring the electrical resistance across it.
• TEER measurements are performed by placing electrodes at both poles of a layer of epithelial cells that have been cultured on a semi-permeable membrane. The electrodes apply an alternating current allowing the measurement of the electrical resistance through the layer of epithelial cells. [0146]
• the TEER ratio is obtained by the following calculation:
• the TEER is measured using an automatic device (REMS). The values are saved and represent our starting value (T0). The cells are then co-incubated in the presence of bacteria (MOI 1/40) for 3 hours, the time that the latter adhere to the cells. Then the cells are challenged with TNF- ⁇ inducing a disruption of the epithelial cell layer and in particular at the tight junctions. 24 hours (T24) after adding the bacteria, the TEER is measured again.
• MERS automatic device
• This figure shows us that the Christensenella minuta bacteria influence the integrity of the intestinal barrier by improving transepithelial resistance, indicating a potential effect on tight junction proteins and a decrease in intestinal permeability.
• the Christensenella minuta bacteria therefore make it possible to restore the resistance of the epithelial membrane.
• the objective of this study is to demonstrate the anti-inflammatory effect in vivo on mice, of bacteria according to the invention.
• the demonstration was carried out on major markers of inflammation, two cytokines: IL6 (interleukin 6) and TNF-a (tumor necrosis factor-alpha).
• IL6 interleukin 6
• TNF-a tumor necrosis factor-alpha
• mice Four week old male C57BL / 6 mice were purchased from Charles River (St Germain sur l'ArbresIe, France).
• mice had a period of acclimatization of one week on the diet based on a standard diet (“chow diet (SafeA04)”) before starting the study.
• Plasma levels of cytokines IL-6 and TNF were determined using ELISA assay kit "Invitrogen ® Luminex ® Magnetic Mouse Cytokine 10-Plex” and according to the manufacturer's instructions. The measurements were carried out by VEBIO (Arcueil, France).
• force-feeding of all groups begins, as well as weight monitoring.
• the control mice ctlr-vehicle and DNBS-vehicle
• the DNBS-Christensenella minuta (C. minuta) mice are force-fed with 150 ⁇ L of C.
• DNBS dinitrobenzensulfate
• the latter is injected directly into the colon of the mouse through a probe introduced into the rectum.
• DNBS is dissolved in 30% ethanol, weakening the intestinal barrier to facilitate entry of DNBS into the tissue, thus triggering the immune system and therefore inflammation.
• the gavages continue during this period, and those of the 5-ASA group begin on the day of this injection at 150 ⁇ L (5-ASA solution prepared every morning, at 2 mg / mouse resuspended in PBS). Euthanasia takes place 3 days after the injection. The so-called “recovery” period is therefore short, but necessary to be able to observe differences with the DNBS control group.
• FIG. 4 represents the various parameters evaluated during a test on an inflammatory model induced by DNBS.
• the macroscopic scores indicate the level of severity of the disease, indicate that following treatments with DSM 22607, a significant decrease in the score was observed, as well as with the pharmacological agent anti-. inflammatory, 5-ASA (5-aminosalicylic acid used as a comparative positive control).
• FIGS. 4C and 4D show us the immunomodulatory potential of DSM 22607, in particular by reducing the presence of neutrophils in the colon, evaluated by following the activity of myeloperoxidase.
• the increase of interleukin-10 in the spleen shows us the anti-inflammatory effects associated with Christensenella minuta.
• mice Three days after the injection, the animals are euthanized. The change in the weight of the mice is measured by taking the weight of the mice daily. During the sacrifice, the colon is opened and cleaned in order to achieve the macroscopic score. The latter is based on the presence of blood in the tissue (hyperemia), the presence of ulcer, intestinal transit (for example if the mouse was constipated) and the thickening of the colon induced by DNBS.
• MPO myeloperoxidase
• the spleen is also recovered during sacrifice (peripheral lymphoid organ) and the cells of the innate and adaptive immune response are stimulated in order to assay the secreted cytokines such as IL-10, anti-inflammatory cytokines.
• cytokines such as IL-10, anti-inflammatory cytokines.
• FIG. 5 represents the effects of other strains of Christensenella minuta during a repetition of the experiment presented in FIG. 4.
• the experimental design and the methods remain similar.
• This experiment teaches us that all the strains of Christensenella minuta tested have the same efficacy in reducing the macroscopic scores following intrarectal injection of DNBS. Likewise, all the strains tested show an anti-inflammatory effect which results in a reduction in the activity of MPO.
• the HT-29 cells were inoculated at a density of 10,000 cells / well in a total volume of 100 ⁇ l in a 96-well plate. After 24 h of incubation, the culture medium was removed from the adherent cells and new medium supplemented with 10% of Christensenella minuta stationary phase supernatant was added (DSMZ, C. minuta 1 and C. minuta 2 and in control GAM (bacterial culture medium)). Each condition was made in 4 replicates. The cells were incubated for an additional 48h or 72h.
• Cell proliferation was determined by the use of the CelITiter-GIo 2.0 assay kit (Promega). The measurements were carried out according to the manufacturer's instructions. Briefly, the plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min and an equal volume of CelITiter-GIo 2.0 reagent was added. directly to the wells (100 mI). The plates were stirred at 300 rpm for 2 min using a rotary shaker, then incubated at room temperature for 10 min. The reaction mixture was then transferred to a white-walled 96-well plate and the luminescent signal was measured using a microplate reader (FLUOstar Omega, BMG Labtech).
• a microplate reader FLUOstar Omega, BMG Labtech
• FIG. 6 represents the influence of strains of Christensenella minuta on the proliferation of tumor cells.
• the inventors observed the influence of strains of Christensenella minuta on the proliferation of tumor cells, we analyzed the effect of these supernatants by treatment of a human colon adenocarcinoma cell line, the HT-29 line. .
• the effect of the supernatants of Christensenella minuta on the proliferation of HT-29 cells was evaluated after 48 and 72 h.
• Akt inhibitor VIII is used as a control for blocking cell proliferation.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Molecular Biology (AREA)
• Rheumatology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Physical Education & Sports Medicine (AREA)
• Dermatology (AREA)
• Immunology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Gastroenterology & Hepatology (AREA)
• Biomedical Technology (AREA)
• Pain & Pain Management (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Virology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (4)

Publications (1)

Family

ID=70110267

Family Applications (1)

Country Status (7)

Families Citing this family (15)

Family Cites Families (3)

• 2020
  • 2020-03-11 WO PCT/EP2020/056565 patent/WO2020182916A1/fr unknown
  • 2020-03-11 US US17/437,492 patent/US20230167401A1/en not_active Abandoned
  • 2020-03-11 BR BR112021017950A patent/BR112021017950A2/pt not_active Application Discontinuation
  • 2020-03-11 EP EP20716170.4A patent/EP3937958A1/de active Pending
  • 2020-03-11 MX MX2021010868A patent/MX2021010868A/es unknown
  • 2020-03-11 JP JP2021553398A patent/JP2022525058A/ja active Pending
• 2021
  • 2021-09-05 IL IL286182A patent/IL286182A/en unknown

Also Published As

Similar Documents

Legal Events