Method and pharmaceutical composition for use in the treatment of nutritional liver diseases or metabolic syndrome

Abstract

The present invention relates to the treatment of nutritional liver diseases or metabolic syndrome. The inventors aimed to identify bacteria through lactic acid bacteria (LAB), such as lactobacillus or bifidobacteria described for their safety used in humans. Up to now, the LAB strains used as probiotics are previously identified for their protective effect on the gut barrier. They isolated LAB strains in patients with excessive and chronic alcohol consumption, showing an absence of advanced liver injury. They focused on LAB strains able to better counteract the harmful effect of enterobacteria isolated from a patient with a severe alcoholic disease. Furthermore, they demonstrated that four bacterial strains specifically improve in vitro gut barrier and / or liver injury both in ALD and MAFLD and also metabolic syndrome in MAFLD, in rodent models. Thus, the present invention relates to specific bacteria and their use in the treatment of nutritional liver diseases or metabolic syndrome.

Description

[0001] METHOD AND PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF NUTRITIONAL LIVER DISEASES OR METABOLIC SYNDROME

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to specific bacteria and their use in the treatment of nutritional liver diseases or metabolic syndrome.

[0004] BACKGROUND OF THE INVENTION:

[0005] Metabolic liver diseases, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are leading causes of chronic liver diseases worldwide with relatively few effective and safe treatments (1-3). ALD is driven by high alcohol consumption, with the risk of liver disease increasing as alcohol consumption rises beyond around 30 g / day: 7.1-fold relative risk at 50 g / day, rising to a 26-fold relative risk at 100 g / day (4). Similarly, NAFLD is driven by dietary calorific excess leading to overweight and obesity associated with multiple features of the metabolic syndrome (central obesity based on the waist circumference of > 80 cm and > 94 cm in women and men, respectively, in European population, elevated fasting blood sugar > 1 g / L (or drug treatment for elevated glucose), elevated triglyceride levels >1.7 mmol / L (or drug treatment for elevated triglycerides), and elevated blood pressure >130 mmHg systolic or >85 mmHg diastolic (or drug treatment for hypertension) (5). Recently, the nomenclature of NAFLD has been updated to MAFLD for the metabolic-associated fatty liver disease and more recently MASLD since even low alcohol consumption could have a synergistic detrimental effect with the presence of metabolic syndrome (6).

[0006] The histological spectrum of liver injury in MAFLD and ALD follows a similar progression from steatosis, inflammation, fibrosis, and cirrhosis until hepatocellular carcinoma. The disease progression depends on many common factors, including dysregulation of fatty acid homeostasis, reactive oxygen species production, mitochondrial dysfunction, or endoplasmic reticulum stress (5, 7). These biological dysfunctions are dependent on environmental factors, nutritional habits, and also the genetic of the host, and some relevant variants of genes mainly involved in lipid metabolism are currently identified (8). However, during the last two decades, a causal role of the intestinal microbiota (IM) in MAFLD and ALD has been well established playing a significant role in the individual susceptibility of patients to develop the disease. While the burden of liver diseases worldwide continues to grow, relatively few effective and safe treatments are available for ALD and MAFLD, including preventing the progression of the disease by manipulating the gut bacteria opens a new field of research to develop efficient treatments.

[0007] In ALD, the inventors have demonstrated that both the sensitivity and the protection from alcohol-induced liver lesions are transmissible from patients to mice by fecal transplantation. They also showed that microbiota modulation by transplanting human microbiota from alcoholic patients that do not develop alcoholic hepatitis could improve alcohol -induced liver lesions in mice (9, 10). The mechanisms by which the IM can induce or conversely protect from alcohol-induced liver injury depends on the gut microbiota function, including the production of metabolites and gut homeostasis. Thus, in patients with ALD or rodent models of ALD, the gut barrier disruption was associated with a decrease in the tight junction proteins expression and mucus and antimicrobial peptides production. This gut homeostasis is improved as well as the liver injury in rodent models of ALD after changes of the IM by fecal microbiota transfer, prebiotics, or probiotics. To improve ALD progression, several probiotics previously studied for their effect on gut barrier homeostasis have been tested in rodent models and human trials. The first use of probiotics in ALD was performed in a rodent model of alcohol intake by using Lactobacillus rhamnosus GG (LGG), which improved gut leakiness and liver inflammation (11-13) adding oat fiber or supernatant of LGG induced similar results, suggesting that bacterial products were partially involved in the protective mechanisms (14-17). More recently, we have shown that Akkermansia muciniphila. which is decreased in ALD patients with a severe stage of the disease, can improve liver injury in a rodent model of ALD (18). VSL#3, a mixture of eight probiotic strains, also improved liver lesions in ALD in humans and rodents (19, 20). Clinical studies were conducted using several probiotics, Bifidobacterium bifidum and L. plantarum 8PA3 slightly increased liver ALT after 5 days of treatment (21). Similarly, a mix of L. rhamnosus R0011 and L. helveticus R0052 versus placebo was used in ALD patients with cirrhosis and alcoholic hepatitis (22). After 7 days of treatment, ALT, gamma-glutamyl transferase, and the Child-Pugh score were improved. However, the main limitation of this study is the concomitant alcohol withdrawal which induced similar improvement of the liver injury, which is not improved by the addition of L. subtilis and Streptococcus faecium (23).

[0008] In MAFLD, the analysis of the IM composition was recently reviewed, showing the difficulty of finding specific gut microbial signatures associated with obesity, T2D, and liver injury progression (nonalcoholic steatohepatitis (NASH) and fibrosis) (24). However, it has also demonstrated that IM has a causal role in MAFLD and metabolic syndrome (25, 26). Obesity is associated with a lowering of IM diversity and a lower count of bacterial genes (27). After surgical treatment of obesity and weight loss, the patients who better improved the associated metabolic syndrome, including T2D and liver injury, are those who better restored the IM diversity (24, 28). Several probiotic strains have been found to be effective in different experimental rodent models of MAFLD. For example, supplementation with LGG reduced steatosis, liver pro-inflammatory cytokines, and restored gut barrier function in fructose- induced MAFLD (29, 30). Similarly, Bacteroides uniformis CECT7771 and Bifidobacterium pseudocatenulatum CECT7765 improved steatosis and immune defense mechanisms in high- fat diet (HFD) fed mice (31, 32), whereas Lactobacillus casei Shirota was shown to protect against NASH induced by a methionine-choline deficient diet (33). Finally, administration of VSL#3, improved liver steatosis and insulin resistance in high fat diet-fed mice (34), and reduced liver inflammation and serum ALT in ob / ob mice (35). Few randomized, prospective clinical trials have been performed in humans to assess the effect of probiotic administration on MAFLD. However, a meta-analysis of four trials involving 134 MAFLD / NASH patients provided evidence that probiotic therapies may be effective in improving NAFLD markers, despite the use of different bacterial strains and administration protocols (36). In particular, the use of probiotics was associated with lower plasma aminotransferase and total cholesterol levels, lower systemic inflammation, and improved insulin resistance. More recently, A. muciniphila was identified and used in an obese rodent model showing mainly an effect on the improvement of T2D (37-40). As a species of bacteria never used as a probiotic in humans, its safety was recently addressed, and then its use in humans (41). A. muciniphila as mentioned above, has also been shown to improve liver injury in an ALD rodent model. However, an increase in the abundance of 4. muciniphila has been associated with a higher risk of developing multiple sclerosis and Parkinson's disease (42-45).

[0009] These encouraging results strongly suggest that the use of probiotics may be an effective addition to the treatment of metabolic liver disease.

[0010] SUMMARY OF THE INVENTION:

[0011] In this study, the inventors aimed to identify bacteria through lactic acid bacteria (LAB), such as lactobacillus or bifidobacteria described for their safety used in humans. Up to now, the LAB strains used as probiotics are previously identified for their protective effect on the gut barrier (46). In this study, they isolated LAB strains in patients with excessive and chronic alcohol consumption (AUD, alcohol use disorder), showing an absence of advanced liver injury. Then, they focused our findings on LAB strains able to better counteract the harmful effect of enterobacteria isolated from a patient with a severe alcoholic disease. Furthermore, they demonstrated that four bacterial strains alone or in combination specifically improve in vitro gut barrier and / or liver injury both in ALD and MAFLD and also metabolic syndrome in MAFLD, in rodent models.

[0012] Thus, the present invention relates to specific bacteria and their use in the treatment of nutritional liver diseases or metabolic syndrome. Particularly, the invention is defined by its claims.

[0013] DETAILED DESCRIPTION OF THE INVENTION:

[0014] Bacteria of the inventions

[0015] The inventors discovered particularly four promising specific bacteria which could be used in the treatment of nutritional liver diseases or a metabolic syndrome.

[0016] Thus, a first object of the invention relates to a Lactobacillus reuteri (Sreu4, L4) deposited in accordance with the Budapest Treaty, on February 21, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5934 or a Lactobacillus reuteri (Sreu5, L5) deposited in accordance with the Budapest Treaty, on February 21, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5935 or a Lactobacillus rhamnosus (Srha47, S47) deposited in accordance with the Budapest Treaty, on February 21, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5936 or a Lactobacillus rhamnosus (Srha5, S5a) deposited in accordance with the Budapest Treaty, on December 13, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-6023.

[0017] According to the invention, the bacteria L4, L5, S5a and S47 are named “the bacteria of the invention”.

[0018] Table 1: bacteria of the invention

[0019] In a particular embodiment, the bacteria of the invention are isolated.

[0020] In a particular embodiment, the bacteria of the invention are in the form of viable cells or non-viable cells. In a particular embodiment, the bacteria of the invention are heat- inactivated bacteria. In a particular embodiment, the invention relates also to the supernatant of the bacteria of the invention or to the metabolites produced by the bacteria of the invention or to a fraction of the bacteria of the invention.

[0021] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L4.

[0022] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L5.

[0023] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lac tobacillus reuteri L5.

[0024] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lac tobacillus rhamnosus S5a.

[0025] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lac tobacillus rhamnosus S5a.

[0026] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L5 and the bacteria lactobacillus rhamnosus S5a.

[0027] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5.

[0028] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L4 and the bacteria Lac tobacillus rhamnosus S5a.

[0029] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L5 and the bacteria Lac tobacillus rhamnosus S5a.

[0030] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4, the bacteria Lac tobacillus reuteri L5 and the bacteria Lac tobacillus rhamnosus S5a. In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L4, the bacteria Lac tobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a.

[0031] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 or the supernatant of the bacteria or the fraction of the bacteria with any other bacteria suitable.

[0032] Methods of treatment and use of the bacteria of the invention

[0033] A second object of the invention relates to a bacteria Lactobacillus rhamnosus or Lactobacillus reuteri or a combination thereof for use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

[0034] According to the invention, nutritional liver diseases regroup the metabolic-associated fatty liver disease (MAFLD), the non-alcoholic fatty liver disease (NAFLD) and the alcoholic liver disease (ALD).

[0035] Thus, the invention also relates to a bacteria Lactobacillus rhamnosus or Lactobacillus reuteri or a combination thereof for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0036] In a particular embodiment, the bacteria Lactobacillus rhamnosus or Lactobacillus reuteri can be the bacteria L4, L5, S5a or S47 of the invention.

[0037] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L4 for use in the treatment of metabolic- associated fatty liver disease (MAFLD) in a subject in need thereof.

[0038] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L5 for use in the treatment of metabolic- associated fatty liver disease (MAFLD) in a subject in need thereof.

[0039] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5 for use in the treatment of metabolic- associated fatty liver disease (MAFLD) in a subject in need thereof.

[0040] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus rhamnosus S5a for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0041] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus rhamnosus S5a for use in the treatment of metabolic- associated fatty liver disease (MAFLD) in a subject in need thereof. In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a for use in the treatment of metabolic- associated fatty liver disease (MAFLD) in a subject in need thereof.

[0042] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5 for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0043] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L4 and the bacteria Lac tobacillus rhamnosus S5a for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0044] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L5 and the bacteria Lac tobacillus rhamnosus S5a for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0045] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus reuteri L4, the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0046] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lac tobacillus reuteri L4, the bacteria Lac tobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a for use in the treatment of metabolic-associated fatty liver disease (MAFLD) in a subject in need thereof.

[0047] In one embodiment, the invention relates to a combination of the bacteria Lactobacillus rhamnosus S47 or the supernatant of the bacteria or the fraction of the bacteria with any other bacteria suitable for the treatment of the present invention.

[0048] As used herein, the terms “Lactobacillus rhamnosus" also known as “Lacticaseibacillus rhamnosus" has its general meaning in the art and denotes a short Gram-positive homofermentative facultative anaerobic non-spore-forming rod that often appears in chains.

[0049] As used herein, the term “Lactobacillus reuteri” also known as “Limosilactobacillus reuteri” has its general meaning in the art and denotes a lactic acid bacterium Gram-positive found in a variety of natural environments, including the gastrointestinal tract of humans and other animals.

[0050] As used herein the term “metabolic-associated fatty liver disease (MAFLD)” previously known has “Non-alcoholic fatty liver disease (NAFLD)” denotes an excessive fat build-up in the liver with metabolic risk abnormalities and therefore regroups non-alcoholic diseases like non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) whether or not there is an associated alcohol consumption. For a good definition of MALFD, see for example the reference 6 or the articles Mohammed Eslam et al., Gastroenterology 2020, PMID: 32044314 and Fouad Y. et al, Liver Int. 2020 Jun;40(6): 1254-1261. According to the invention, NAFLD can also refers to MASLD for “Metabolic dysfunction-Associated Liver Disease” (see for example: https: / / www.aasld.org / new-masld-nomenclature).

[0051] As used herein, the term “Alcoholic liver disease (ALD)” denotes liver lesions related to excessive alcohol consumption and encompasses fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis.

[0052] As used herein, the term “metabolic syndrome” denotes a syndrome that regroups at least three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL) (see for example: www.nhlbi.nih.gov / health / metabolic-syndrome). Metabolic syndrome is associated with the risk of developing cardiovascular disease and type 2 diabetes.

[0053] In a particular embodiment, the bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or the bacteria of the invention can be used as probiotic bacteria.

[0054] As used herein, the term “probiotic bacteria” denotes bacteria which are ingested live in adequate quantities and can exert beneficial effects on human health. They are now widely used as a food additive for their health-promoting effects. Most of the probiotic bacteria are Lactic Acid Bacterium (LAB) and among them, strains of the genera Lactobacillus and Bifidobacteria are the most widely used probiotic bacteria.

[0055] As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0056] As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject according to the invention is a human. More particularly, the subject is suffering from nutritional liver diseases or metabolic syndrome.

[0057] Accordingly, in a particular embodiment the present invention relates to a method of treating nutritional liver diseases or metabolic syndrome in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a bacteria Lactobacillus rhamnosus or a Lactobacillus reuteri or a combination thereof.

[0058] Therapeutic composition

[0059] Bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or bacteria of the invention may be administered in the form of a pharmaceutical composition or of a formulation, as defined below. In another aspect, the invention relates to a therapeutic composition comprising a bacteria Lac tobacillus rhamnosus or Lactobacillus reuteri or a combination thereof for use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

[0060] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L4.

[0061] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L5.

[0062] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5.

[0063] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47 and the bacteria Lactobacillus rhamnosus S5a.

[0064] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus rhamnosus S5a.

[0065] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a.

[0066] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5.

[0067] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus rhamnosus S5a.

[0068] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a.

[0069] In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus reuteri L4, the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a. In one embodiment, the invention relates to a therapeutic composition comprising a combination of the bacteria Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4, the bacteria Lactobacillus reuteri L5 and the bacteria Lactobacillus rhamnosus S5a.

[0070] In one embodiment, the invention relates to a a composition comprising a combination of the bacteria Lactobacillus rhamnosus S47 or the supernatant of the bacteria or the fraction of the bacteria with any other bacteria suitable.

[0071] As used herein, the term “formulation” relates to the compositions or group of one or several ingredients, i.e., class and a number of the elements present in a complex substance (food product or dosage form, among others) and proportion in which they are found.

[0072] According to the invention, bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or the bacteria of the invention or the therapeutic composition of the invention are administrated in a therapeutically effective amount.

[0073] Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. For example, the bacteria of the invention can be combined with a food supplement.

[0074] As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or preventive result. A therapeutically effective amount the bacteria of the invention or of the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the bacteria of the invention or of the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the bacteria of the invention or of the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the bacteria of the invention or for the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician or pharmacist having ordinary skill in the art may readily determine and prescribe the effective amount of the bacteria of the invention or of the composition of the invention required. For example, the physician could start doses of the bacteria of the invention or of the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the inhibitor or the composition of the present invention will be that amount of the inhibitor which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, and for example, the ability of the bacteria of the invention or the composition of the invention to treat nutritional liver diseases or metabolic syndrome may, for example, be evaluated in an animal model of steatosis, NASH or fibrosis. One of the ordinary skills in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of bacteria of the present invention is about 106CFU / g (Colony-forming unit), 107CFU / g, 108CFU / g, 109CFU / g or IO10CFU / g. Administration may e.g. be oral, intranasal or rectal. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments. An effective dose of bacteria of the present invention may also be administered using a daily, weekly, biweekly, or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks, or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of bacteria of the present invention in an amount of about 106CFU / g (Colony-forming unit), 107CFU / g, 108CFU / g, 109CFU / g or IO10CFU / g using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0075] Administration may be oral, intranasal or rectal. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic or preventive situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms- An effective dose of bacteria of the present invention may also be administered using a daily, weekly, biweekly, or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of bacteria of the present invention in an amount of about 106CFU / g (Colonyforming unit), 107CFU / g, 108CFU / g, 109CFU / g or IO10CFU / g. The treatment can last between 2 and 6 weeks, particularly 1 month.

[0076] In other words, the quantity of bacteria or population of bacteria of the invention or composition of the invention administered to a subject in need thereof is between 106CFU / g (Colony-forming unit), 107CFU / g, 108CFU / g, 109CFU / g or IO10CFU / g. The bacteria or the population of bacteria of the invention can be administrated 1, 2, 3, 4, or 5 times to the subject in need thereof during between 2 and 6 weeks and particularly during 1 month.

[0077] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0078] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.

[0079] The pharmaceutical compositions of the invention can be formulated for an oral, intranasal or rectal administration.

[0080] In particular, the bacteria of the invention may be administrated with physiological serum or can be freeze-dried.

[0081] In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids (like Syrup, Spray, capsules, gums, tablet) for oral administration; time release capsules; and any other form currently can be used.

[0082] In each of the embodiments of the treatment methods described herein, the bacteria or the composition of the invention of the invention are delivered in a manner consistent with conventional methodologies associated with the management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the bacteria of the invention or of the composition of the invention are administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[0083] According to the invention, the formulation according to the invention is for use in diets and particularly for use in infant and / or adult and / or special diets.

[0084] According to the invention, the formulation according to the invention is for use in the preparation of food supplements.

[0085] According to the invention, the formulation according to the invention is for use in the preparation of special formulas for oral and / or enteral nutrition.

[0086] Combination and kit of part

[0087] In another aspect of the invention, the bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent or other treatment. The present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent or treatment.

[0088] According to the invention, anti -nutritional liver diseases agents or anti-metabolic syndrome agents may be added to the pharmaceutical composition or used in combination with the bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or bacteria of the invention in the case of the treatment of MAFLD or ALD.

[0089] Anti-metabolic liver disease or anti-MAFLD agents or anti-metabolic liver disease or anti-MAFLD treatment include for example liver transplantation, diet modifications, drug that target insulin-resistance, overweight and obesity (GLP-1 agonists, GIP agonists, SGLT2 inhibitors, DPP4 inhibitors, metformin, PPAR agonists), bile acids (ursodeoxycholic acid, obeticholic acid) and more generally, all drugs that target nutritional liver diseases.

[0090] Another aspect of the present invention relates to i) a bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or a combination thereof, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

[0091] As used herein, the term “simultaneous use” denotes the use of bacteria Lactobacillus rhamnosus o Lactobacillus reuteri of the invention or bacteria of the invention and at least one therapeutic active agent occurring at the same time.

[0092] As used herein, the term “separate use” denotes the use of bacteria Lactobacillus rhamnosus ox Lactobacillus reuteri of the invention or bacteria of the invention and at least one therapeutic active agent not occurring at the same time.

[0093] As used herein, the term “sequential use” denotes the use of bacteria Lactobacillus rhamnosus ox Lactobacillus reuteri of the invention or bacteria of the invention and at least one therapeutic active agent occurring by following order.

[0094] Particulary, the bacteria of the invention are the bacteria L4, L5, S5a and S47.

[0095] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0096] FIGURES:

[0097] Figure 1: Isolation of LAB strains from noAH patient decreasing enterobacteria growth. (A) Screening diagram for the LAB strains from the patient. (B) Inhibition of sAH enterobacteria by supernatant, heat-inactivated, or living LAB strains decreasing enterobacteria growth by at least 1.5 log. The sAH enterobacteria treated with D / L lactic acid were used as a control in statistical analysis. Three parallel experiments were performed for each group.

[0098] Figure 2: Decrease of sAH enterobacteria cell-entrance in Caco2 cells. Caco2 / TC7 monolayer cells cultured were infected with sAH enterobacteria in presence of LAB strain supernatant (A), heat-inactivated bacteria (B) or living bacteria (C). Experiments were done in Caco2 / TC7 cells treated with alcohol or not. Three parallel experiments were performed for each group.

[0099] Figure 3: Measurement of IL8 production after sAH enterobacteria infection and LAB treatment of Caco2 cell line. Caco2 / TC7 monolayer cells cultured were infected with sAH enterobacteria in the presence of LAB strain supernatant (A), heat-inactivated bacteria (B), or living bacteria (C). IL8 production was quantified in the supernatant recovered after the infection by sAH enterobacteria for each condition. Experiments were done in Caco2 / TC7 cells treated with alcohol or not. Three parallel experiments were performed for each group. Figure 4: Decrease of sAH enterobacteria translocation through an in vitro epithelium treated with LAB strains. In Caco2 / TC7 monolayer cells cultured in a transwell system in the presence of alcohol, sAH enterobacteria infections were done in the presence of LAB strain supernatant (A), heat-inactivated bacteria (B), or living bacteria (C). (D) Inhibition of sAH enterobacteria by living S5a and S5b. Three parallel experiments were performed for each group.

[0100] Figure 5: Isolated LAB strains decrease liver injury in alcohol-fed mice. (A) Plasma alanine aminotransferase (ALT) level. (B) Liver mRNA levels of pro-inflammatory cytokines and chemokines. (C) Quantification of Oil-Red-O staining of the liver using Image J software. (D) Liver mRNA levels of genes involved in fibrosis. (E) Liver mRNA levels of genes involved in lipid metabolism.

[0101] Figure 6: Isolated LAB strains slightly improve gut injury in alcohol-fed mice. (A) mRNA levels of genes relative to the gut barrier disruption in the Small intestine (SI) (left panel) and colon (right panel). (B) Quantification of Oil-Red-O staining of the liver using Image J software. (C) mRNA levels of genes relative to mucus production in SI (left panel) and colon (right panel).

[0102] Figure 7: Isolated LAB strains improve liver steatosis and metabolic syndrome in obese mice. (A) Oral glucose tolerance test (OGTT) after 2 months of treatment with LAB strains, significant results compared to PBS for L4 (&), S47 / L4 (*). (B) Insulin tolerance test (ITT) after 2 months of treatment with LAB strains had significant results compared to PBS for L5 (&), S47 / L5 (*). (C) Quantification of liver triglycerides. (D) Plasma alanine aminotransferase (ALT) level.

[0103] Figure 8: Isolated LAB strains decrease liver injury compared to LGG strain in obese mice. Liver mRNA levels of (A) pro-inflammatory cytokines and chemokines, (B) genes involved in fibrosis.

[0104] Figure 9: Effect of isolated LAB strains in liver metabolism. Liver mRNA levels of genes involved in lipid metabolism including de novo lipogenesis (A) or bile acid metabolism (B).

[0105] Figure 10: Histological sections of brown adipose tissue and molecular markers of lipid metabolism in adipose tissues. (A) Representative hematoxylin-and-eosin-stained pictures of BAT (scale bar, 250 pm) and quantification of the lipid content for each group of mice. (B) Liver mRNA levels of genes involved in lipid metabolism. Figure 11: Isolated 3 LAB strains improve metabolic syndrome in obese mice. (A)

[0106] Insulin tolerance test (ITT) after 7 weeks of treatment with LAB strains had significant results compared to PBS (*). (B) Ratio of the protein expression for pAkt / Akt in liver tissue. Table 2: Summary of the in vitro effect of LAB strains. EXAMPLE:

[0107] Material & Methods

[0108] Patients.

[0109] Two patients with alcohol use disorder (AUD) were included in the study (data not shown). One patient with severe alcoholic hepatitis (sAH) showed a Maddrey score > 32 and a liver biopsy with a histological score of AH > 6 with neutrophilic infiltration. The second patient has no advanced liver injury (noAH). Patients were admitted to the hepato- gastroenterology department of Antoine-Beclere University Hospital, Clamart, France. Alcoholic patients consumed more than 50 g of alcohol per day over the previous year, were negative for hepatitis B surface antigens, and seronegative for antibodies against hepatitis C virus (HCV). They did not show any of the exclusion criteria: gastrointestinal bleeding, bacterial infection, hepatocellular carcinoma or other carcinomas, acute pancreatitis, other severe associated disease, diabetes mellitus, dyslipidemia, presence of anti-HIV antibodies, and antibiotic or probiotic intake in the last 3 months. The study was carried out in accordance with the Helsinki Declaration and was approved by the He de France VII ethics committee (Bicetre Hospital, 94270 le Kremlin-Bicetre, France). All the participants provided written informed consent.

[0110] Recovery of stools and isolation of LAB strains from feces sampling.

[0111] Feces have been recovered and immediately stored at 4 °C in an anaerobiosis generator (Genbox, Biomerieux, Capronne, France) to favour the preservation of anaerobic bacteria. The feces were frozen at -80°C directly. Fecal bacteria were isolated by plating on lactic acid bacteria- selective Man Rogosa Sharp (MRS) broth (Gibco, Thermo Fisher Scientific) at 37°C under aerobic and anaerobic conditions for 24h. For isolation of strains, successive dilutions and spreads were carried out from each dilution tube to obtain isolated strains on MRS agar plates. After 48 hours of incubation at 37°C, the different strains obtained on a dish were put back into culture separately in MRS broth. All of the isolated strains were frozen at -80°C for preservation in 25% glycerol (bacterial pellet / glycerol 4v / lv).

[0112] Culture of isolated LAB strains for experiments.

[0113] LAB strains were cultured in MRS medium in aerobe or anaerobe conditions. MRS Broth contains plant peptone (Gibco,) or animal peptone (Gibco, Thermo Fisher Scientific) were used. Modified MRS broth was prepared as previously described (48). LAB strains, including LGG, are cultured in aerobe conditions during 24h at 37°C in MRS. The culture of LAB strains used to treat mice was specifically counted using the microbial cell counter Quantom Tx (Logos Biosystem). Sequencing of LAB strain.

[0114] Total sequencing of isolated LAB strains was performed using nanopore technology (GridlON, Life&Soft, Fontenay aux Roses). ORF and sequences alignments were performed using QIAGEN CLC Genomics Workbench.

[0115] Isolation of enterobacteria from a sAH patient.

[0116] Isolation of a pool of enterobacteria from feces of sAH patient was previously described (47). Briefly, fecal bacteria were cultured in Luria-Bertani Broth Miller (LB) (Gibco, Thermo Fisher Scientific) at 37°C under aerobic conditions and, stored with 25% glycerol at -80°C until experiments. For each experiment, a culture of the enterobacteria pool was carried out in LB broth. After 24 h of culture at 37°C, 100 pL of this bacteria culture was used for cell culture experiments.

[0117] Inhibition of the enterobacteria growth by LAB strains or their supernatant.

[0118] LAB strains were cultured in MRS liquid medium at 37 C for 24h, centrifuged at 4000 rpm / min for 10 min at 4°C. The centrifugated supernatant was passed through a sterile 0.22pm PES filter unit (Pall, Fischer Scientific; France). Next, we added 500 pl of the LAB strain supernatant to 500 pl of the sAH enterobacteria pool and incubated at 37°C for 24h in LB broth. Controls were conducted in similar conditions of culture: sAH enterobacteria alone (500pl of sAH pool with 500pl of IX PBS) and sAH enterobacteria in the presence of a mix of D-L lactic acid 25mM, respectively (500pl of sAH pool with 500pl of MRS-DL lactic acid at 50 mM- Acros Organics) to reproduce a pH value of the medium ranging from 4.3 to 4.5. After 24h of incubation, aliquots were recovered, serially diluted, and plated on LB agar to determine the bacterial colony counts. Three parallel experiments were performed for each group.

[0119] Double-Layer Agar (DLA) Method.

[0120] The anti-microbial properties of LAB strained were checked following freezing, heating, and enzymatic treatments by the double-layer agar method (49). Firstly, the sAH enterobacteria pool was cultured as above, then mixed in surfusion 3% LB Miller agar medium (Gibco, Fisher Scientic, France), and the mixture was poured on plates as the first layer of agar. After the plates cooled, we used a 5 mm punch to drill holes in them. LAB strains (109cfu / mL) treated by different treatments were plated to each hole. The second layer of 6% LB Miller agar was poured on top of the first layer, and plates were incubated at 37 C for 24 h. The inhibition diameter corresponding to the anti-microbial effect was measured and compared to the antimicrobial effect of the strain without treatment. LAB strains were treated by heat (inactivation at 100°C), freezing / thawing (5 cycles of freezing / thawing at -20°C) and digestive enzymes Catalase (600pg / mL) (MP Biomedicals, 190311), Alpha-amylase (200pg / mL) (MP Biomedicals, 100447), Papain (200pg / mL) (Roche Diagnostics, 10108014001), Lactate Dehydrogenase (250pg / mL)(Merck, 59747), Trypsin (200pg / mL), (Gibco, 25300-054), Proteinase K (lOOpg / mL - Fisher Bioreagents, BP1700-50), Pepsin from porcine gastric mucosa (200pg / mL - Sigma, P7000) and Peroxidase (200pg / mL - Thermo Fisher, Chem Lab CL00.2301.5000). All assays were performed in triplicate.

[0121] Quantification of lactic acid and IL8.

[0122] Lactic acid enantiomers produced by LAB strains were quantified using D and L-lactic acid dosage kits (R-biopharma, Roche). The concentration of each enantiomer was determined in the supernatant of LAB strains cultured for 24h at 37°C in MRS broth. IL8 quantification was performed using an IL8 assay kit (Human IL8 ELISA kit, Diaclone). Three experiments were performed for each lactic acid and IL8 quantification.

[0123] Caco2 / TC7 cell line culture and exposure to LAB strains.

[0124] The human colon adenocarcinoma Caco-2 / TC7 cell line clone was grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM, Gibco, Fisher Scientific) (25mM glucose) supplemented with 15% heat-inactivated (30 minutes, 56°C) fetal calf serum and 1% non-essential amino acids during 14 days (Gibco, Thermo Fisher Scientific). The non-alcohol condition was similarly cultured 7 days more (Day 21), and a chronic alcohol condition was exposed to EtOH 50mM from day 14 to day 21 as previously described (47). Experiments were carried out at 37° C in a 10% CO2 / and 90% air.

[0125] Inhibition of enterobacteria cell entry in Caco-2 / TC-7 cells.

[0126] The Caco-2 / TC-7 cells were grown in 24 well culture plates (Costar, Dutscher, France). Prior to infection, cell monolayers were washed twice with PBS. sAH enterobacteria (108bacteria / mL) pool resuspended in the culture medium were added to each well of the tissue culture plate. LAB strains supernatant, or the heat-inactivated LAB strains were added to the medium at the same time as sAH enterobacteria. Then, the plates were incubated at 37°C for 3 hours and washed three times with sterile PBS. After infection, the supernatant was collected for further IL8 quantification. Then monolayers were washed twice with sterile PBS, and extracellular bacteria were killed by treatment with gentamicin (Img / mL, Ih, 37°C). After washing twice with sterile PBS, cells were lysed with cold water for 30 min at +4°C. Appropriate dilutions were plated on LB broth agar to determine the number of viable intracellular bacteria. After 24h of incubation at 37°C, forming bacterial colonies were counted. Each assay was conducted in triplicate with three successive passages of Caco-2 / TC-7 cells.

[0127] To determine the effect of living LAB strains on the enterobacteria cell-entry, infections were performed as above, except that living LAB strains were added 2 hours after the sAH infection and during the last hour of sAH enterobacteria infection to avoid disruption of the cell monolayer.

[0128] Inhibition of enterobacteria translocation through Caco-2 / TC-7 cells translocation.

[0129] Caco-2 / TC7 cells were cultured for 21 days in a 12-wells Transwell system with 4 pm- pore size polycarbonate membranes (Corning™ Transwell™ Multiple Well Plate, Dutcher) that delineate an apical (luminal) reservoir and a basolateral reservoir. Infections with sAH enterobacteria and LAB strains treatments were performed as described above. Viable translocated bacteria were recovered in the lower chamber of the transwell system after eight hours of infection. Appropriate serial dilutions were done and plated onto LB broth agar plates. After 24h of incubation at 37°C, forming bacterial colonies were counted. Each assay was conducted in triplicate with three successive passages of Caco-2 / TC-7 cells.

[0130] Mice and diets.

[0131] Seven- week-old female C57BL / 6J mice were purchased from Janvier laboratory (Le Genest, France) for chronic alcohol exposure. Animals were kept in humidity- and temperature- controlled rooms on a 12-hour light-dark cycle and had access to a chow diet and water ad libitum until they weighed at least 20g. Then, mice were fed a liquid diet adapted from Lieber DeCarli (LDC) for 21 days, as previously described based on the NIAAA model 3, but without a binge administration of alcohol at the end 10. Briefly, the ethanol diet was obtained by adding absolute ethanol to a solution of LDC powder (Ssniff, Spezialdiaten GmbH, Soest, Germany) in filtered water. After seven days of adaptation to the animal facility and seven days of adaptation to the semi-liquid diet, mice were given increasing amounts of ethanol for seven days (1% increase every two days). The final ethanol concentration in this liquid diet was 5% (vol / vol); ethanol accounted for 28% of the total caloric intake. Alcohol-fed groups were allowed free access to the 5% (vol / vol) ethanol diet for seven days. The control diet was obtained by replacing the ethanol with an isocaloric amount of maltodextrin (Maldex 170, Safe, France). LAB strain mixture was given by gavage daily from the addition of 4% alcohol to the diet until the end of the protocol. During the LDC diet, all mice did not have access to drinking water. Diet consumptions were recorded and were similar between the alcohol -fed groups (data not shown). Five-week-old male C57BL / 6J mice were purchased from Janvier laboratory (Le Genest, France) to induce obesity and nonalcoholic liver disease. Mice are maintained under a 12h light / dark schedule, with food and water ad libitum, and treated in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Mice were fed a high-fat, high sucrose, and cholesterol 1% diet (HFSC) (SNIFF, Germany). Mice were weighed weekly, and diet intake was measured and averaged for each cage (3 mice per cage). LAB strain mixture was given by gavage after eleven weeks of HFSC diet, three times a week for ten weeks, and until the end of the protocol (Fig. 7 to 10) or LAB strain mixture was given by gavage after nine weeks of HFSC diet, three times a week for 8 weeks, and until the end of the protocol, (Fig. 11). The French Ministry specified housing conditions regarding the protection of laboratory animals (agreement number C-92-023-01). All mouse experiments were approved by and performed in accordance with the guidelines of the local ethics committee and approved by the French Ministry, 2020020314244050 vl (APAFIS#23907).

[0132] Glucose and insulin tolerance tests.

[0133] An oral glucose tolerance test (OGTT) was performed as follows: a glucose load (2 g / kg) was given by gavage after six hours of fasting. Then, glucose concentration was determined in blood obtained from a small incision in the mouse tail and pressed on a glucometer strip at 0, 15, 30, 60, 90, and 120 min post-gavage (Accu-Chek® Performa - Roche, Switzerland). The glycemia curve and the area under the glucose-time curve were calculated. Insulin tolerance test (ITT) was performed as follows: animals were fasted for six hours to receive insulin (0.6 mU / g of mouse body weight) via gavage. Serum glucose concentrations were measured and calculated as above. OGTT was performed after eight weeks of HFSC diet and after the LAB strains treatments, the week before mice were euthanized. ITT was done two days before the mice were euthanized.

[0134] Tissues and samples.

[0135] Mice were anesthetized, and blood samples were collected in EDTA-coated tubes. The serum was used for liver alanine aminotransferase (ALT), aspartate aminotransferase (AST), HDL-cholesterol, and triglycerides (TG) determination. The livers were excised, weighed, and either fixed in 4% paraformaldehyde or frozen for further analysis. A liver sample was frozen in a tissue-freezing medium (TFM, Microm-Microtech France). The small intestine (SI) proximal ileum and large intestine (LI) colon were cut into two pieces: flushed, opened longitudinally, cut into 2 cm sections, and fixed in 4% paraformaldehyde, and the other was frozen for further RNA extraction. Brown adipose tissue (BAT) and white adipose tissue (WAT) were cut into two parts: one piece was fixed in 4% paraformaldehyde for histology investigations, and the other was frozen for further RNA extraction. All samples were stored at -80°C until use. The caecal content was collected and frozen in two separate tubes and frozen.

[0136] Measurement of liver triglycerides and plasmatic transaminases.

[0137] Triglycerides were extracted using an Abeam Triglyceride Assay Kit - Quantification (Cambridge, UK) and measured with a Mithras LB940 (Berthold Technologies). The level of TG is expressed in nmol per milligram of the liver. Plasma transaminases (ALT and AST), cholesterol, HDL, and triglycerides were assessed using an automatic analyzer AU400 (Olympus Diagnostics, Rungis, France).

[0138] Tissues histology.

[0139] Liver, WAT, BAT, SI, and colon paraffin sections (3 pm thick) were stained with hematoxylin and eosin (HE). Colon paraffin sections (3 pm thick) were stained with Alcian blue to evaluate the mucus production. Frozen liver sections (7 pm thick) were used for Oil- Red-0 staining using standard procedures. All images were digitally captured from the scan slides using NanoZoomer 2.0-RS and the NDP view2 software (Hamamatsu, Japan). All image quantification analysis were performed using the FIJI software (https: / / imagej.net / Fiji) 50, 51. For Oil-Red-O staining area measurement, we have developed an image processing macro using conversion in RGB Stack and thresholding to define the region of interest (staining or fluorescence) and the « Freehand selection » tool to determine the total area of interest. For alcian blue slides / mucus quantification, we have specifically developed an image processing macro using a “freehand selection” tool to determine the region of interest (tissue) and its area; then, color deconvolution and thresholding on the color channel of interest (here blue) with defined values to limit measure to the staining of interest (mucus). For HE staining and lipid droplet / adipocytes analysis (number, mean diameter, area), we used the previously validated FIJI plugin: Adiposoft 52. For HE staining to evaluate lipid droplets amount, we have specifically developed an image processing macro using, among others, thresholding to compare the area represented by lipids (white region / unstained) to the total area of tissue in the field. For each image analyzes, values were exported to Excel and Prism-GraphPad for statistical analysis and graphical representations.

[0140] RNA extraction and quantification.

[0141] Mice livers, BAT, and WAT, were disrupted in the Qiazol solution. Total RNA was extracted using a Qiagen RNeasy Lipid Tissue Mini Kit (Courtaboeuf, France). The ileum and colon were disrupted with an MP Biomedicals FastPrep and extracted using Qiagen RNeasy Tissue Mini Kit (Courtaboeuf, France). The RNA integrity number (RIN) was determined using an Agilent Bioanalyzer 2100 system with the RNA 6000 Nano Labchip kit. The samples used had a RIN of 8 for liver tissues and a RIN of 7 for the gut, BAT, and WAT tissues. Five pg of each total RNA sample was reverse transcribed for cDNA synthesis. A 6 pl mix containing five pg of RNA, random hexamers (Roche Diagnostics, Meylan, France), and 10 mM dNTP Mix (Invitrogen, Carlsbad, CA) was prepared for each sample. Mixtures were heated at 65°C for 5 min and cooled on ice. Then an 8 pl reaction mix containing 1 pl M-MuLv RT (Invitrogen), 4 pl 5x Buffer (Invitrogen), 2 pl 0.1 M dithiothreitol (Invitrogen), and 1 pl Protector RNase Inhibitor (40 U / pl; Invitrogen) was added. The reaction conditions were 10 min at 25°C, 50 min at 50°C, and 15 min at 70°C.

[0142] Gene expression analysis by quantitative PCR.

[0143] Real-time qPCR was performed in a Light Cycler 480 (Roche Diagnostics) using the LC FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Amplification was initiated with an enzyme activation step at 95°C for 10 min, followed by 40 cycles consisting of a 20- sec denaturation step at 95°C, a 15-sec annealing step at the appropriate temperature for each primer, and a 10-sec elongation step at 72°C. Data were analyzed using LC 480 Software (Roche Diagnostics). Relative gene expression was normalized to the HPRT or GAPDH reference gene. Fold induction was calculated using the control group as standard, and data were shown in fold induction. Primer sequences of the amplified targets were generated including Gapdh and Hprtl used as housekeeping genes.

[0144] Protein extraction and Western blot analysis.

[0145] Liver proteins were extracted from 30mg of tissue using a RIP A buffer. Proteins (30 pg) were subjected to SDS-PAGE analysis on 4-12% gels and transferred to PVDF (polyvinylidene difluoride) membranes. Polyclonal mouse P actin (Sigma-Aldrich A2066) were used as loading controls. Rabbit polyclonal for total Akt, phospho-Akt (Ser473 and Thr308) were purchased from Cell Signaling (ref 4058 and 2603). To estimate the levels of phosphorylation of Akt reported relative to actin, densitometry was performed using VisionWorks LS software.

[0146] Statistical analysis.

[0147] Results are shown as mean ± SEM. The ANOVA or the nonparametric Kruskall-Wallis test with Tukey or Dunn’s multiple comparison posthoc test were used to compare the means of groups, as appropriate (Graphpad Prism 9.3, Graphpad Software Inc, La Jolla, California, USA); p < 0.05 was considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.

[0148] Results

[0149] Isolation of lactic acid bacterial strains from AUD patients without liver injury.

[0150] We isolated several lactic acid bacteria (LAB) from the stools of a patient with excessive consumption of alcohol as defined by EASL guidelines and showing an absence of liver injury and named noAH (data not shown). The stools were cultured in MRS broth to favor the growth of LAB strains in aerobe and anaerobe conditions, and we isolated 37 and 75 strains, respectively (Figure 1 A). Then, we determined the ability of LAB strains to inhibit the growth of a pool of enterobacteria isolated from a patient with severe alcoholic hepatitis (sAH). To this end, the stools of the sAH patient were grown in LB broth to favor the growth of enterobacteria identified by sequencing (data not shown). Isolation of this pool of enterobacteria was previously described (47).

[0151] The supernatant of an overnight culture of the 112 LAB strains was incubated with the sAH pool for 3h, 6h, or 24h. 21 and 61 LAB strains showed a anti-microbial effect in aerobe and anaerobe conditions, respectively (data not shown). To overcome that lactic acid produced by LAB strains could be responsive to the antimicrobial effect, the control condition was treated with 50mM lactic acid in all experiments. Among these 112 LAB strains, we specifically focused on S5 and S47 for their high anti-microbial effect, whatever the incubation time. L4 and L5 strains were selected both for their anti-microbial effect but also for their morphological differences. Then, we addressed the anti-microbial effect of the heat-inactivated and living bacteria and compared them to the effect of the supernatant (Figure IB). Only 4 strains showed an inhibitory effect greater than 1.5 log whether culture supernatant, heat-inactivated, or living bacteria were used (Figure IB). The production of D and L-lactic acid enantiomers was quantified and compared with the ratio observed for Lactobacillus rhamnosus GG (LGG) cultures in the same conditions (data not shown). This is consistent with using 50mM of lactic acid to treat the sAH group serving as a control.

[0152] Inhibition of sAH enterobacteria cell-entrance and translocation by LAB strains.

[0153] To go further on the potential beneficial effect of these LAB strains, we used the colon adenocarcinoma Caco-2 / TC7 cell line in culture. These cells are a useful tool to study the protective effect of our isolated LAB strains to counteract the infection by enterobacteria of enterocytes (data not shown). We specifically addressed the protective role of LAB strains on enterobacteria cell entry and enterobacteria translocation. To this end, we used differentiated and polarized Caco2 / TC7 in the presence of alcohol or not. The sAH enterobacteria infected Caco2 / TC7 cells, and co-incubated with LAB strains supernatant, heat-inactivated or living bacteria (Fig. 2A, B, and C). Using the supernatant of LAB strains, a significant decrease of sAH enterobacteria cell entrance was observed, and the highest effect was obtained using S47 supernatant independently of the alcohol exposure (Fig. 2A). Heat-inactivated and living bacteria also induced a significant decrease of the sAH enterobacteria cell entrance (Fig. 2B and C). Of note, S47 allowed the highest reduction of sAH enterobacteria using heat-inactivated or living bacteria in the presence of alcohol or not. Conversely, the LAB strain L5 could not efficiently decrease sAH enterobacteria invasion. Interestingly, the living LAB strain L4 induced the highest decrease of sAH enterobacteria invasion (Fig.2C). The measurement of the IL8 production by Caco2 / TC7 infected by sAH enterobacteria cells was performed. Alcohol exposure majored cell injury, as shown by the higher level of IL8 in the presence of alcohol (Fig. 3). Consequently, LAB strains had a moderate inhibitory effect in this condition, except for the LAB strain S47 and the living S5. The supernatant, heat-inactivated or living LAB strains decreased IL8 production in Caco2 / TC7 cells without alcohol exposure, except the heat- inactivated L5 strain (Fig. 3). In Caco2 / TC7 cells exposed to alcohol, the decrease of IL8 production was mainly observed using living LAB strains.

[0154] Enterobacteria can translocate through an epithelium by invading cells or by a transepithelial pathway. Using monolayers cell in transwell cultures, we evaluated the translocation of sAH enterobacteria, whatever the path. In those conditions, heat-inactivated and living LAB strains did not show a decrease of the translocation (Fig. 4). However, there was a significant decrease of the translocated sAH enterobacteria using LAB strain supernatant except for the L4 strain (Fig.4A).

[0155] Importantly, we evaluated the effect of LGG in all these experiments. We showed that our isolated LAB strains similarly impact the sAH enterobacteria virulence against an in vitro cell epithelium (Fig. 1, 2, 3, 4).

[0156] Identification of isolated LAB strains.

[0157] To translate the relevance of these strains for therapeutic use in preclinical models of MAF and NAFLD, we checked some properties to validate a possible industrial process. These strains were resistant to freezing / thawing and a panel of digestive enzymes (data not shown, see materials and methods). LAB strains were sequenced by nanopore technology and specifically identified (data not shown). Surprisingly, the S5 strain was a mixture of 2 strains. However, we isolated the two strains, S5a referred to L. rhamnosus S5 and S5b referred to L. reuteri S5b and demonstrated that S5a alone mediated the anti-microbial effect (Fig. 4D).

[0158] Finally, based on the effect of strains on sAH enterobacteria virulence and species identification summarized in Table 2, we combine a mix of L. rhamnosus S47 and L. reuteri L4 (S47 / L4) to test its efficiency in the preclinical rodent model of ALD.

[0159] The probiotic mix S47 / L4 improves alcohol-induced liver injury in mice.

[0160] We investigated the protective effect of a mix of two LAB strains, L4 and S47, in alcohol-induced liver injury by feeding C57BL / 6J mice with alcohol. The effects of the LAB Strains S47, L4, and the S47 / L4 mix were compared to a control group receiving PBS and a group receiving LGG. All treatments improve liver steatosis (Fig. 5). The level of transaminases was not modified by treatments, including in the LGG-treated group of mice. However, the mRNA expression of several cytokines and chemokines showed a decrease in the inflammatory process in mice treated with the mix S47 / L4. Evaluation of fibrosis by quantifying genes involved in collagen renewal showed that S47 / L4 improves fibrosis as LGG.

[0161] Concerning the effect of LAB strains on the gut barrier, we found that the mix S47 / L4 improves mucus production as LGG. The effect on the production of antimicrobial peptides and tight junction protein did not show clear differences, and LGG induced similar effects (Fig.6).

[0162] Effect of LAB strains in diet-induced obesity mice

[0163] We investigated the protective effect of LAB strains alone or in combination in obese mice by feeding C57BL / 6J mice with high fat, high sucrose, and 1% cholesterol diet (HFSC). After 8 weeks of the HFSC diet, mice were treated with LAB strains three times a week for 8 weeks. Control groups received PBS or LGG. There was no difference in the weight gain of mice nor in the ratio of liver / body weight and WAT / body weight in all groups (data not shown). We addressed insulin resistance in mice through an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT). There was no improvement of insulin resistance in LGG-treated mice, nor in S47-treated mice (Fig. 7A). Conversely, we found an improvement in glucose tolerance in mice treated with L4 and S47 / L4 and in insulin tolerance in L5 and S47 / L5 (Fig. 7A and B). The improvement of insulin resistance is associated with an improvement in liver steatosis in the four groups of mice, with the highest effect in mice receiving the mix S47 / L5 (Fig. 7C). ALT was improved in mice receiving the LAB strain L5 and in a lower extent the S47 and the mix S45 / L5 (individual p values 0.0099, 0.054 and 0.056 respectively). In the liver, there was a decrease in the inflammatory profile of mRNA-expressing cytokines and cytokines (Fig 8A and B). The analysis of genes expression involved in the liver metabolism showed that S47, L4 and the mix S47 / L5 decrease FAS gene expression conversely of the LAB strains (Fig. 9 A). The gene expression of SREBP1 was increased by our LAB strains conversely to LGG (Fig. 9A). The analysis of other genes expression involved in the lipid metabolism did not show any significant modification (Fig. 9B).

[0164] Peripheric insulin resistance was addressed by analyzing adipose tissue homeostasis. We did not find either difference in the weight of WAT regarding the treatments or in histological morphology regarding the number and size of lipid droplets (data not shown). Conversely, we found modifications of BAT morphology at the level of histological examination with a specific decrease in the size of lipid droplets suggesting an activation of the metabolic function of WAT (Fig 10A). We observed a decrease in the FAS mRNA expression whatever the bacterial strain used without significant modification in the expression of mRNA nor for CPT1 neither UCP1 suggesting (Fig 10B). In a second in vivo experimental procedure with a shorter treatment, we found a tendency for the mix L4 / L5 to decrease liver steatosis. However, the presence of S47 seems to be linked to an improvement of the lix efficiency as we confirmed that the insulin-resistance was improved by the mix of the 3 LAB strains, S47, L4 and L5, as shown by the ITT test after 7 weeks of treatment (Fig. 11 A) correlated to the activation of the insulin receptor signaling pathway as shown by the increase of the Akt phosphorylation (Fig. 1 IB).

[0165] Conclusion :

[0166] In this study, the inventors showed that Lactobacillus rhamnosus and Lactobacillus reuteri, alone or in combination can be used in the treatment of nutritional liver diseases or metabolic syndrome. Particularly, they discovered 4 different strains (L4, L5, S5a and S47) which can be used alone or in combination in the treatment of nutritional liver diseases or metabolic syndrome. They showed that these strains can improve liver function better than the strain LGG.

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Claims

CLAIMS:

1. A Lactobacillus reuteri (Sreu4, L4) deposited in accordance with the Budapest Treaty, on February 21, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5934 or a Lactobacillus reuteri (Sreu5, L5) deposited in accordance with the Budapest Treaty, on February 21, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5935 or a Lactobacillus rhamnosus (Srha47, S47) deposited in accordance with the Budapest Treaty, on February 21 , 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-5936 or a Lactobacillus rhamnosus (Srha5a, S5a) deposited in accordance with the Budapest Treaty, on December 13, 2023 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-6023.

2. A bacteria Lactobacillus rhamnosus or Lactobacillus reuteri or a combination thereof for use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

3. A supernatant or a fraction of the bacteria according to the claim 1.

4. A i) bacteria Lactobacillus rhamnosus or Lactobacillus reuteri of the invention or a combination thereof, and ii) at least one further therapeutic active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

5. A therapeutic composition comprising a bacteria Lactobacillus rhamnosus or a Lactobacillus reuteri or a combination thereof for use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

6. A therapeutic composition comprising the supernatant or the fraction according to the claim 3 for use in the treatment of nutritional liver diseases or metabolic syndrome in a subject in need thereof.

7. A method of treating nutritional liver diseases or metabolic syndrome in a subject in need thereof comprising administering to the subject a therapeutically effective amountof a bacteria Lactobacillus rhamnosus or a Lactobacillus reuteri or a combination thereof.

8. A method of treating nutritional liver diseases or metabolic syndrome in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of the supernatant or the fraction according to the claim 3.

9. The bacteria or combination according to the claims 2 or 4 or the supernatant or the fraction according to the claim 3 or the therapeutic composition according to the claim 5 or 6, or the method according to the claim 7 or 8 wherein the bacteria Lactobacillus rhamnosus o Lactobacillus reuteri are the bacteria L4, L5, S47 or S5a according to the claim 1.

10. The bacteria for use according to the claims 2 to 9 wherein the bacteria are S47 and L5.

11. The combination for use according to the claims 2 or 4 or the combination for the composition according to the claims 5 or 6 or the combination for the method according to the claims 7 or 8 wherein the bacteria combined are the Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L4.

12. The combination for use according to the claims 2 or 4 or the combination for the composition according to the claims 5 or 6 or the combination for the method according to the claims 7 or 8 wherein the bacteria combined are the Lactobacillus rhamnosus S47 and the bacteria Lactobacillus reuteri L5.

13. The combination for use according to claims 2 or 4 or the combination for the composition according to claims 5 or 6 or the combination for the method according to claims 7 or 8 wherein the bacteria combined are the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5.

14. The combination for use according to the claims 2 or 4 or the combination for the composition according to the claims 5 or 6 or the combination for the method according to the claims 7 or 8 wherein the bacteria combined are the Lactobacillus rhamnosus S47, the bacteria Lactobacillus reuteri L4 and the bacteria Lactobacillus reuteri L5.

15. The bacteria or combination according to the claims 2, 4, 10, 11, 12, 13 or 14 or the therapeutic composition according to the claims 5, 6, 11, 12, 13 or 14 or the methodaccording to the claims 7, 8, 11, 12, 13 or 14 wherein the nutritional liver diseases is the metabolic-associated fatty liver disease (MAFLD), the non-alcoholic fatty liver disease (NAFLD) or the alcoholic liver disease (ALD).

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