Use of amoebae for the treatment of metabolic syndrome

Abstract

Metabolic syndrome (MS) is a clustering of several human conditions including abdominal obesity, high blood pressure, dyslipidemia, and high blood sugar, all being risk factors of type 2 diabetes, cardiovascular disease, and metabolic-associated fatty liver disease (MAFLD). By its prevalence and its consequences, MS is a major public health problem worldwide. It is a direct consequence of modern Western lifestyle including changes of dietary pattern. Here the inventors hypothesize that the loss of protozoa that have ever inhabited our intestine may have also contributed to MS outbreak. To test this hypothesis, mice were colonized by Entamoeba muris and then fed a high fat diet. Colonized animals had more limited dysbiosis and less secondary bile acids in their intestine with less interleukin 1β and lipid accumulation in their liver. Thus, the present invention relates to the use of amoebae for the treatment of metabolic syndrome and in particular metabolic-associated fatty liver disease (MAFLD).

Description

[0001] USE OF AMOEBAE FOR THE TREATMENT OF METABOLIC SYNDROME

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular microbiology, obesity and hepatology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Human morbid conditions like insulin resistance, obesity, Metabolic dysfunction-associated fatty liver disease (MAFLD), dyslipidemia and hypertension (altogether referred as metabolic syndrome, or MS) are among the leading causes of type 2 diabetes and cardiovascular diseases which are themselves major causes of death and disability in developed as well as developing countries.1,2The Westernized dietary pattern characterized by high intakes of saturated fats, refined grains, salt and sugars with low intake of fruits and vegetables is a recognized risk factor for MS (for review see3). Food components carry direct metabolic effects but they also contribute to these conditions through changes in the gut microbiota composition.4Among these changes, a lower bacterial diversity with a lower relative abundance of Firmicutes (newly renamed as Bacillota) are commonly reported.5

[0006] While modem Western lifestyle has led to major dietary changes, many other aspects have been impacted such as hygiene, comfort, medical advances, clothing, leisure activities or travels. Because all these changes are closely interconnected, joint risk factors associated with a westernized way of life may have been neglected. Among these potential non dietary risk factors, we question here the impact of a reduced exposure to intestinal eukaryotes.

[0007] The mammalian intestinal tract is hosting eukaryotes including macroparasites like helminths and single-celled organisms like protozoa.6,7Eukaryotic microbes are generally considered to be parasites. This is the case for helminths and several protozoa like Giardia lamblia, Entamoeba histolytica, and Cryptosporidium spp. which contribute to well-defined diseases. At the opposite, protozoa like Blastocystis hominis, Dientamoeba fragilis or Entamoeba spp. (other than E. histolytica') have no or questionable pathogenicity.7They are present across various healthy human populations in the world and recent studies retrieved them even more frequently in healthy controls than in patients with digestive symptoms or diseases like inflammatory bowel disease.7,8These findings, and the co-evolution over millions of years of protozoa and their hosts, argue for a definition of their mode of interaction as commensalism rather than parasitism.9Consequently, their recent disappearance from the human digestive tract under better hygiene conditions in westernized populations could be detrimental.

[0008] Among protozoa, B. hominis and D. Fragilis remains frequent in the western world. At the opposite and despite a worldwide distribution, commensal amoebae (mainly Entamoeba coli. E. dispar and E. Hartamni) have become rare in westernized populations while they remain common in rural developing countries.6,10,11Therefore, they are good candidates to investigate the role of protozoa on MS. Unlike Entamoeba hisloliliccF1. Entamoeba coli and other commensal species have no clear pathogenic effect.7On the contrary, they have been associated with beneficial changes in the gut bacterial component of the microbiome, including increased diversity and higher levels of bacteria belonging to Firmicutes (Bacillota) phylum11,13,14,15,16which may then counteract the effect of the westernized diet.

[0009] To examine the hypothesis of an effect of gut colonization by commensal amoebas on MS, we investigated the consequences of infesting mice with amoeba Entamoeba muris (E. muris). This amoeba is closely related to Entamoeba coli which colonizes the human digestive tract.17It is recognized as non-pathogenic, even in immunocompromised animals reared under standard conditions.

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention is defined by the claims. In particular, the present invention relates to the use of amoebae for the treatment of metabolic syndrome.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Metabolic syndrome (MS) is a clustering of several human conditions including abdominal obesity, high blood pressure, dyslipidemia, and high blood sugar, all being risk factors of type 2 diabetes, cardiovascular disease, and metabolic-associated fatty liver disease (MAFLD). By its prevalence and its consequences, MS is a major public health problem worldwide. It is a direct consequence of modem Western lifestyle including changes of dietary pattern. Here the inventors hypothesize that the loss of protozoa that have ever inhabited our intestine may have also contributed to MS outbreak. To test this hypothesis, mice were colonized by Entamoeba muris and then fed a high fat diet. Colonized animals had more limited dysbiosis and less secondary bile acids in their intestine with less interleukin 10 and lipid accumulation in their liver. These findings strongly argue for a protective effect of amoebas on metabolic syndrome, in particular MAFLD.

[0014] Accordingly, the present invention relates to a method of treating metabolic syndrome in a subject in need thereof comprising administering a therapeutically effective amount of amoebae.

[0015] As used herein, the term “patient” or “subject” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the patient is a human. In some embodiments, the patient is a human who is susceptible to having a metabolic-associated fatty liver disease.

[0016] As used herein, the term “metabolic syndrome” includes the co-occurrence in an adult subject of several metabolic risk factors, including at least three of the following five traits: abdominal obesity, which can be, for example, a waist circumference in men of greater than or equal to 90 cm and in women greater than or equal to 80 cm; elevated serum triglycerides, which can be, for example, greater than or equal to 150 mg / dL, or drug treatment for elevated triglycerides; reduced serum HDL cholesterol level, which can be, for example, below 40 mg / dL in men and below 50 mg / dL in women, or drug treatment for low HDL cholesterol; hypertension, which can be, for example, systolic blood pressure greater than 130 mmHg and diastolic blood pressure greater than 85 mmHg, or drug treatment for hypertension; and elevated fasting plasma glucose, which can be, for example, greater than or equal to 100 mg / dL, drug treatment for elevated glucose, or previously diagnosed type 2 diabetes.

[0017] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including the treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient 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 patient 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 patient during the treatment of an illness, e.g., to keep the patient 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 particular predetermined criteria [e.g., disease manifestation, etc.]).

[0018] In some embodiments, the subject is overweight, i.e. a subject having a BMI index superior to 25kg / m2. As used herein, the term “BMI” or “body mass index” has its general meaning in the art and refers to the ratio, which is calculated as body weight per height in meter squared (kg / m2). The BMI provides a simple means of assessing how much an individual's body weight departs from what is normal or desirable for a person of his or her height. Common definitions of BMI categories are as follows: starvation: BMI — less than 15 kg / m2; underweight — BMI less than 18.5 kg / m2; ideal — BMI from 18.5 to 25 kg / m2; overweight — BMI from 25 to 30 kg / m2; obese — BMI from 30 to 40 kg / m2; morbidly obese — BMI greater than 40 kg / m2. While simple, the BMI method of characterizing the body weight property of a person is not always correct. For example, the BMI does not take into account factors such as frame size, muscularity or varying proportions of e.g. bone, cartilage, and water weight among individuals. Thus, the accuracy of BMI in relation to actual levels of body fat mass may be distorted by such factors as fitness level, muscle mass, bone structure, gender, and ethnicity. Also, people with short stature and old people tend to have lower BMI values. It is considered, however, that the skilled person, e.g. a physician, will be able to take these factors into account when making the BMI assessment of any given individual.

[0019] In particular, the subject is obese or is at risk of obesity. As used herein, the term “obesity” refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg / m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg / m2. An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg / m2or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg / m2. A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg / m2to less than 30 kg / m2or a subject with at least one co- morbidity with a BMI of 25 kg / m2to less than 27 kg / m2. The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian -Pacific countries, including Japan, "obesity" refers to a condition whereby a subject has a BMI greater than or equal to 25 kg / m2. An "obese subject" in these countries refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m2. In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg / m2 to less than 25 kg / m2.

[0020] In some aspects, the method of the present invention is particularly suitable for the treatment of metabolic-associated fatty liver disease (MAFLD).

[0021] As used herein, the term “metabolic-associated fatty liver disease” or “MAFLD” has its general meaning in the art and encompasses all fatty liver disease states, which aligns with the traditional view that non-alcoholic fatty liver disease (NAFLD) represents a spectrum of liver disease associated with insulin resistance, starting with pure “benign” steatosis (NAFL), through to non-alcoholic steatohepatitis (“NASH”), which is the inflammatory state that can lead to advanced fibrosis or cirrhosis. Non-alcoholic fatty liver disease (NAFLD) represents one of the most recurrent and severe pathologies, especially among obese and diabetic patients, yet a specific therapy is far from being available. NAFLD is defined as the accumulation of fat in the liver, but not as secondary consequence of alcohol consumption. The term "NASH", as used herein, collectively refers to the state where the liver develops a hepatic disorder (e.g., inflammation, ballooning, fibrosis, cirrhosis, or cancer), or the state where the liver may induce such a pathological condition, and "NASH" is distinguished from "simple steatosis"; i.e., a condition in which fat is simply accumulated in the liver, and which does not progress to another hepatic-disorder-developing condition.

[0022] In some aspects, the method of the present invention is also particularly suitable for reducing hepatic steatosis. As used herein, the term "hepatic steatosis" refers to the condition in which fat accumulates in liver tissue, heart muscle tissue or other muscle tissues.

[0023] As used herein, the term "amoeba" refers to unicellular eukaryotic microorganisms found in natural aquatic and terrestrial environments under temperate climate, but also under more extreme environments such as polar melt water, arid land or tropical forest. These protists move and feed by emitting cytoplasmic extensions called pseudopods. While some are parasitic, such as Entamoeba histolytica, others who do not depend on a host are qualified as “Free-living”. In the environment, amoebae graze naturally on bacteria, fungi or other protists that they engulf by phagocytosis into digestive vacuoles. Thus, amoebae are predators that naturally regulate populations of multiple microorganisms in the environment and play an important ecological role. Amoebae useful in the present invention can be identified and isolated using technique well known in the Art, including SSU-rDNA sequencing (Small subunit ribosomal DNA) by PCR analyses. Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy -based analyses.

[0024] According to the present invention, the amoeba is not a parasitic amoeba. As used herein, the term “parasitic amoeba” refers to an amoeba that injures or damages tissue or causes other forms of disease in mammals. Thus, according to the present invention, the amoeba is commensal. As used herein, the term "commensal" refers to a microorganism that is nonpathogenic to a host and is part of the normal microflora of the host.

[0025] In some embodiments, the amoeba of the present invention is Entamoeba coli. As used herein, the term “Entamoeba coi has its general meaning in the art and is a non-pathogenic species of Entamoeba that exists as a commensal microorganism in the human gastrointestinal tract.

[0026] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the active agent to be effective, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient in need thereof will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0027] The active agents (i.e. amoebae) are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art. The pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example, as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference. The present pharmaceutical formulations comprise active agent (i.e. amoebae)s (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations of the present invention can also comprise active agent (i.e. amoebae)s which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like. Pharmaceutical compositions of the present invention can also comprise conventional pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality-adjusting agents, buffers, and pH-adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the present invention can be packaged for use in liquid form or can be lyophilized.

[0028] In some embodiments, the active agent (i.e. amoebae) of the present invention is administered locally to the gastrointestinal tract of the subject. In some embodiments, the active agent (i.e. amoebae) is administered via oral ingestion. Typically, the amoeba are administered to the patient in the form of cysts. The effective amount of the active agent (i.e. amoebae) can be locally administered to the colon of the patient by oral ingestion of a unit dosage form such as a pill, tablet, or capsule, comprising an effective amount of the active agent (i.e. amoebae) which is enterically coated so as to be released from the unit dosage form in the lower intestinal tract, e.g., in the ileum and in the colon of the patient. Enteric coatings remain intact in the stomach, but will dissolve and release the contents of the dosage form once it reaches the region where the pH is optimal for dissolution of the coating used. The purpose of an enteric coating is to substantially delay the release of the active agent (i.e. amoebae) until it reaches its target site of action in the ileum or colon. In particular, a useful enteric coating is one that remains intact in the low pH environment of the stomach, but readily dissolved when the optimum dissolution pH of the particular coating is reached. This can vary between pH 3 to 7.5 depending upon the chemical composition of the enteric coating. The thickness of the coating will depend upon the solubility characteristics of the coating material and the site to be treated. The most extensively used polymer for enteric coating is cellulose acetate phthalate (CAP). However, CAP has an optimum dissolution pH greater than 6, thus early drug release may occur. Another useful polymer is polyvinyl acetate phthalate (PVAP) which is less permeable to moisture and gastric fluid, more stable to hydrolysis and able to dissolve at a lower pH, which could also result in early release of the active agent (i.e. amoebae) in the duodenum.

[0029] 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.

[0030] FIGURES:

[0031] Figure 1. Entamoeba muris reduces hepatic steatosis. Study of the fatty liver disease in C57B1 / 6 mice fed with high fat diet (HFD) and colonized with / out Entamoeba muris (E. muris). (a) Microscopic examination of hepatic steatosis after mice livers were paraffin-embedded, sectioned, and stained with H&E (left column) and contrasted with Oil Red O (right column) for the visualization of lipids. Bar represents 100 pm. (b) Histological score of the relative lipid accumulation based on Oil red O staining. Hepatic (c) triglyceride and (d) cholesterol amounts in the liver, (e) mRNA expression levels of various proteins related with the fatty acid P- oxidation and de novo lipogenesis. (f) Correlation analysis between the relative abundance of E. muris and the concentration of cyclic adenosine monophosphate (cAMP) in the cecum. Data are presented as mean ± SEM. Statistical analyses were performed using the Mann- Whitney U test. Significant differences / correlations were recorded as *p<0.05 and ****p<0.0001.

[0032] EXAMPLE:

[0033] Material & Methods

[0034] E. muris'. In vitro culture, cyst purification, microscopic observation and PCR methods.

[0035] E. muris was cultured in vitro and cryopreserved according to Kobayashi et al.17Trophozoites and cysts were counted under a light microscope (x20 and x60 objectives, department of parasitology, Bichat hospital, Paris) at 24-hour intervals for 5 days. On day 5, cysts were centrifuged (5 min. at 275G), rinsed with distilled water and treated with 0.05 N chloridric acid for 10 min. in order to retain only cysts resistant to gastric passage. They were then counted and resuspended in water at a concentration of 1,000 cysts / 100 pL. Microscopic observation of E. muris was performed in the feces or fecal content of mice (with a drop of water on an object slide) during follow-up and at the end of the experiments. M.I.F. (Merthiolate, Iodine, Formol) staining was also performed to better visualize cytoplasm and nuclear structures.

[0036] The presence of E. muris was also monitored by PCR and Real-Time PCR. In brief, DNA was extracted from mouse feces frozen at -80°C using the QIAamp Fast DNA Stool Mini Kit, according to the manufacturer's recommendations (Qiagen). Mechanical lysis with FastPrep (MP Biomedicals) was added to the protocol prior to thermal lysis at 95°C. Specific amplification of E. muris 18S RNA gene region was performed using the following primers, (suppl. Table 1). Real-time polymerase chain reaction was carried out with SYBR Green (Qiagen) using the LightCycler® 480 (Roche).

[0037] Mouse experiments and analytical procedures.

[0038] Five-week-old wild-type (WT) C57B1 / 6 male mice (Janvier, France) were acclimatized for 1- week prior to experiments and kept under Specific Opportunist Pathogen Free (SOPF) conditions. Experiments were approved by the institutional animal care and use committee (APAFIS# 12939-201801081625584, Paris, France). Mice were inoculated by gavage with 1,000 cysts for the experimental group or with 100 pL of sterile water for the control group. The high-fat diet (SAFE®, 246 HF) was started in mice nine days after gavage. The percentage of calories provided by lipids was 45.5%. The “normal diet” groups received a maintenance diet (suppl. table 2). For 70 days, the animals were weighed twice a week, and fresh feces were collected and immediately frozen to study the microbiota and to monitor the presence of the amoeba by PCR. After 70 days, mice were euthanized by cervical dislocation. Blood and samples were collected for further analysis, as detailed below.

[0039] For glucose tolerance test, mice were gavaged with glucose at a dose of 2.0 g / kg body weight, after 16 hours of fasting. Tail blood glucose concentrations were measured at 0, 5,15, 30, 60, 90 and 120 min. One week later, mice were and then intraperitoneally injected with insulin (ITT) at a dose of lU / kg body weight after four hours of fasting. Tail blood glucose concentrations were measured at 0, 5,15, 30, 60, 90 and 120 minutes.

[0040] Blood glucose values were determined using an ACCU-Check glucose monitor (Roche Diagnostic Inc.). Serum HDL cholesterol, triglycerides (TG), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) concentrations were determined using an automated Monarch device (Biochemistry Laboratory, Faculte de Medecine Bichat, France). Hepatic TGs and cholesterol were extracted with the Folch procedure and measured with a colorimetric kit according to the manufacturer’s instructions (Diasys).

[0041] Samples of the liver tissue were fixed in 4% paraformaldehyde, embedded in paraffin, cut, and stained with hematoxylin and eosin (H&E). Slides were assessed blind by an experienced pathologist. To detect fat deposition in the liver, frozen sections were rinsed with distilled water, stained with Oil-Red O. Morphometry software was used to quantify the percentage of Oil red O-positive cells, as well as the number and total surface area of lipid vacuoles on HE.

[0042] 16S rRNA Microbiome sequencing

[0043] DNA was extracted from cecal samples using a QIAamp PowerFecalPro DNA kit (Qiagen, Hilden, MD), following manufacturer’s instructions. Twenty -two DNA extracts were quantified on a Qubit4 fluorimeter using the dsDNA HS Assay Kit (Life Technologies; USA) and stored at -20°C before further analyses. DNA samples were sequenced after a two-step PCR library preparation, according to the recommendations of the 16S Metagenomic Sequencing Library Preparation Guide (Illumina, San Diego, CA, USA). Briefly, for the first PCR reaction, the V4 hyper-variable region of the 16S rDNA was amplified using the primers 515-F: 5’ - TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GTG CCA GCM GCC GCG GTAA -3’ (SEQ ID NO: 1) and 806-R: 5’ - GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG AC A GGG ACT ACH VGG GTW TCT AAT-3’ (SEQ ID NO: 2). Amplicons were then purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN). A second PCR reaction was performed to incorporate a sample-specific barcode, using a Nextera XT index kit (Illumina, USA). After amplicons purification, DNA concentration was controlled by qPCR using the KAPA Library Quantification kit (Roche). A master DNA pool was then generated in equimolar ratios. PhiX Control v3 (Illumina) was added to check the quality of the run. The pooled product quantity was controlled on a Qubit4 fluorimeter, loaded into an Illumina MiSeq cartridge, and sequenced on an IlluminaMiSeq sequencer (paired-end reads, 2 x 300 bp).

[0044] At the end of the run, FastQ files were generated and quality control was performed. Sequences were demultiplexed and paired-end amplicon reads were processed using the FROGS 3.2 pipeline (Find Rapidly OTU with Galaxy Solution), with the Galaxy platform (https: / / galaxy.migale.inra.fr / ).48Briefly, forward and reverse reads were trimmed for adaptor and PCR primers removal, merged, and chimeric sequences were removed. Reads were then clustered in operational taxonomic units (OTUs) and filtered with a minimum relative abundance threshold of 0.005%.

[0045] Taxonomic assignment was performed against the 16S SILVA 138 pintail 100 databases. Before analysis of a and P diversities and microbiota composition, all samples were rarefied to the same depth, with a minimum reads number of 25,000. Alpha-diversity within group was estimated using the Shannon diversity index. Beta-diversity between groups was evaluated by calculating Bray-Curtis distances between samples. Ordination using principal coordinates analysis (PcoA) was performed to represent biodiversity distribution at the OTU level between groups. Lastly, differential biomarkers between various groups were observed using LEfSe algorithm (linear discriminant analysis coupled with effect size)49

[0046] Untargeted metabolomics analysis

[0047] The analysis of metabolites was carried out by the pharmacology and immunoanalysis department (SPI) of the Joliot Institute at the CEA in Saclay using the LC-HRMS (Liquid Chromatography -High Resolution Mass Spectrometry). Metabolites were extracted from 10 mg aliquots of lyophilized cecal contents. Untargeted metabolomics experiments were performed by LC-HRMS using a combination of two complementary chromatographic methods, consisting of reversed-phase chromatography (Cl 8 chromatographic column) and hydrophilic interaction chromatography (HILIC) for the analysis of hydrophobic and polar metabolites, respectively. An internal standard solution was added to all the samples in order to check the consistency of the analytical results in terms of signal and retention time stability throughout the experiment. Additionally, a quality control sample was obtained by pooling 20pL of each sample preparation. It was injected every 10 samples to evaluate the analysis error for each metabolite. Data extraction was performed by the platform. Compounds were annotated using public and internal databases (SPI). Mass spectrometry-based metabolite levels were calculated based on the area under the peak curve, which allows comparative analysis between groups but not absolute quantification.

[0048] RNA isolation, quantitative Real-Time PCR assays and cytokine analyses.

[0049] Total RNAs was extracted from frozen tissues using NucleoSpin RNA kit according to manufacturer’s instructions (Macherey Nagel). RNA concentration and purity were determined by NanoDrop (Thermo Scientific). For reverse transcription of RNA into cDNA, High-Capacity cDNA Reverse Transcription Kit was employed. Real-time polymerase chain reaction was carried out with SYBR Green (Qiagen) using the LightCycler® 480 (Roche). Changes in mRNA expression were determined by calculating the fold-changes using the comparative threshold cycle (Ct) method. Primer sequences are listed in Supplementary Table 3.

[0050] U-PLEX kits developed by the Mesoscale Discovery were used to assess the levels of some cytokines in the serum and tissue. IL-ip, IL-6, IL-12, TNF-a, IFN-y, TGF-pi, IL-10 and IL-7 were detectable. For blood analysis, serum was undiluted. For tissue analysis, proteins were extracted from frozen organs using an ULTRA TURRAX and lysis buffer with protease and phosphatase inhibitors cocktail. Protein concentration was evaluated by Braford protein assay (Bio-Rad). For TGF-pi, samples were acid-treated and base-neutralized prior to execution of the standard Mesoscale Discovery Soluble protein assessment protocols using the U-plex kit. Cytokine levels were measured from 25 pL of samples or standard. Plates were run on the Mesoscale Discovery instrument (Mesoscale Discovery) and cytokine levels calculated using a standard curve of known cytokine quantities.

[0051] Statistical analysis

[0052] GraphPad Prism 9 (GraphPad Software, La Jolla, CA) was used for statistical comparisons. All data are presented as mean ± SEM. Differences between two groups were assessed by an unpaired two-tailed Student's t-test (normal distribution) or by a two-tailed Mann-Whitney test (non-normal distribution). In the case of more than two groups, significance was analyzed by one-way ANOVA with Bonferroni multiple comparison test (normal distribution) or Kruskal- Wallis test with Dunn-Bonferroni test (non-normal distribution). For statistical analysis of microbiota data, the significance of beta diversity measures was assessed by the PERMANOVA (Permutational Multivariate Analysis of Variance Using Distance Matrices) test. For bacterial abundance, alpha diversity and other data, ANOVA or the Kruskall-Wallis test, with multiple comparisons, were performed. A p-value < 0.05 was considered significant (*p < 0.05; **p< 0.01; ***p< 0.001; ****p < 0.0001) for all analyses, with the exception of the LEfSE algorithm for which the p-value was set at 0.01.

[0053] Results:

[0054] E. m rts can be cultured in vitro, engrafted in vivo, and detected by qPCR in the microbiota of colonized mice

[0055] E. muris was cultured in vitro using the protocol reported by Kobayashi et al.11The reproductive (trophozoites) and infective (cysts) forms of the parasite could be identified (data not shown). As expected, the study of the population dynamics of these two stages revealed a shift in the relative abundances, with cysts increasing in prevalence while the trophozoites decreased (data not shown). C57B1 / 6 mice were inoculated with 1,000 cysts grown in vitro. Cysts were identified in the cecum and feces of these mice from the 7thday after administration until the end of animal’s follow-up (data not shown). Cysts recovered from fecal samples gave rise to mobile forms of parasite (data not shown).

[0056] In order to more easily and accurately detect the presence of the parasites in feces and cecal contents (including the cyst and the trophozoite forms), a qPCR based on E. muris sequence was developed (data not shown). This qPCR was further used throughout our study to monitor the parasites’ presence in colonized mice and its absence in control mice. Additionally, qPCR analyses showed stable amounts of E.muris 70 days after inoculation (data not shown) confirming the long-term engraftment in the digestive tract.

[0057] E. muris restores the prevotellaceae / desulfovibrionaceae ratio of the cecal microbiota, which is reduced in mice fed with high-fat diet.

[0058] Since the parasite is resident in the cecum, we focused on A. / iw / v.s-induced microbiota changes in this organ. After a 70-day inoculation period, we compared four groups of eight mice either fed with normal diet (ND) or high-fat diet (HFD) and infested or not with E. muris. High- throughput sequencing of 16S rDNA revealed that diet had a much greater impact on the microbiota composition than the presence of the parasite, both in terms of alpha (data not shown) and beta diversity (data not shown). As expected, we observed a dysbiosis in the HFD groups compared to the ND groups, which was characterized by a lower alpha diversity (data not shown) and a greater relative abundance of desulfovibrionaceae together with a lower abundance of Prevotellaceae (data not shown). Although ND-fed mice colonized with amoebas had no alterations on the alpha or beta diversities (data not shown), their cecal microbial composition was modified, in particular with an increase in Prevotellaceae (data not shown). Remarkably, in HFD-fed mice, the presence of E. muris significantly reduced HFD- associated anomalies (data not shown) by increasing Prevotellaceae (data not shown) while reducing Desulfovibrionaceae (data not shown). These data were confirmed by a second independent experiment (data not shown). Thus, we concluded that the presence of E. muris restored the Prevotellaceae! Desulfovibrionaceae ratio in the cecum of HFD-fed mice (data not shown).

[0059] E. muris partially restored bile acids changes induced by HFD.

[0060] Given the known impact of the microbiota on the composition of bile acid (BA)18, we analyzed the concentrations of BA in mouse feces by mass spectroscopy. Under ND, the presence of amoeba did not significantly alter the concentrations of primary (data not shown) or secondary (data not shown) fecal BA. In line with previous studies18, HFD significantly increased the levels of primary and secondary BA. In these mice, the presence of E. muris did not alter the total amount of BA but significantly reduced the level of secondary BA, including deoxy cholic acid (DCA) and tauro-deoxycholic acid (TDCA). In the cecum, this reduction affected lithocholic acid (LCA), DCA and TDCA (data not shown), showing a global effect on the three BA transformation pathways (data not shown). The levels of unconjugated BA, which deconjugation is also linked to the microbiota, were decreased (data not shown). Moreover, the BA capable of activating the famesoid X receptor (FXR) was reduced (data not shown). All these changes were negatively correlated with the Prevotellaceae / Desulfovibrionaceae ratio in the caecum suggesting a direct relationship between the microbiota and the BA composition (data not shown). Taken together, these data suggest that the presence of amoebae has the potential to partially restore the levels of secondary and unconjugated BA altered by HFD.

[0061] E. muris reduces the hepatic concentration of ILip in mice on a high-fat diet. Unconjugated BA have a well-known pro-inflammatory effect.18Their lower concentration in HFD-fed mice colonized with E. muris suggested a potential protective effect against inflammation. To test this hypothesis, we assessed the tissue concentrations of inflammation related cytokines, including interleukin 10 (IL 10), interleukin 6 (IL6), interleukin 10 (IL 10), interleukin 12 (IL12), tumor necrosis factor alpha (TNFa), transforming growth factor beta (TGF0), and interferon gamma (IFNy) in the cecum, colon, liver, and mesenteric fat. As expected, HFD induced pro-inflammatory cytokines in several tissues (data not shown). The colonization with E. muris did not restore these changes with the exception of IL10 levels in liver (data not shown). Interestingly, hepatic IL10 amounts strongly correlated with the presence of unconjugated BA (fig 4c). We also observed a correlation between IL10 levels in liver and the concentration of FXR agonist BA data not shown). These results suggest that the presence of amoebae mitigated the pro-inflammatory impact of HFD specifically in liver.

[0062] E. muris reduces the liver steatosis related to high fat diet.

[0063] Because HFD has been associated with MAFLD, we sought for the protective effect of amoeba in liver tissue of HFD-fed mice. Hepatic steatosis -consequence of HFD- was reduced in E. muris colonized mice compared with non-colonized mice (Figure la). Histological observations were confirmed with the measurement of the total area of intracellular vacuoles (data not shown) and fat areas stained with Oil red O (Figure lb). The concentrations of triglycerides (Figure 1c) and cholesterol (Figure Id) in liver were consistent with less fat accumulation. The study of gene expression involved in 0-oxidation and lipogenesis indicated that the downregulation in lipogenesis, reflected by an average of two-fold lower expression, explained the reduced accumulation of fat in the liver (Figure le). Of note, these changes were not associated with changes in blood aspartate aminotransferase (ASAT) or alanine aminotransferase (ALAT) levels (data not shown).

[0064] The examination of other components of MS showed a trend towards a decrease in circulating HDL cholesterol (data not shown) but this association did not reach statistical significance. A non-significant trend towards improvement was also observed for body weight gain (data not shown), body fat composition (data not shown), and white fat percentage (data not shown), but not brown fat (data not shown). No effect was identified on glucose metabolism, either for fasting blood glucose (data not shown), orally induced hyperglycemia (data not shown), or blood glucose response to insulin (data not shown). Overall, the main effect of the presence of E. muris on MS was seen in the liver.

[0065] Finally and because telluric amoebas are able to produce cyclic adenosine monophosphate (cAMP)19and Wang et al. previously reported that cAMP decreases the hepatic steatosis in HFD-fed mice20, we looked for cAMP in our metabolomic dataset. Cecal concentrations of cAMP correlated well with the relative abundance of E. muris (Figure le).

[0066] In summary, a set of inter-related correlations was observed in HFD- fed mice linking E. muris abundance, cAMP concentration, the Prevolellaceael Desulfovibrionaceae ratio in cecum, and secondary BA levels in feces, with IL10 concentration, lipid accumulation and lipogenesis in liver (data not shown).

[0067] Discussion:

[0068] Experiments carried out on HFD-fed mice showed that gut colonization by E. muris had a beneficial impact, marked by a partially reverted dysbiosis and changes in BA metabolism together with a decrease in inflammation and hepatic steatosis.

[0069] In humans, HFD has been consistently associated with a reduction in microbial diversity and changes in microbiota composition.21,22In accordance with the literrature18, our HFD mice model presented a reduced microbial diversity, a decrease in Prevotellaceae, and an increase in Desulfovibrionaceae .

[0070] In HFD-fed mice, the presence of amoebae was associated with notable changes in the microbiota, particularly characterized with an increase of Prevotellaceae and a decrease of Desulfovibrionaceae . Thus, E. muris colonization ameliorated the bacterial dysbiosis induced by diet. While previous studies have associated the presence of amoebae with greater richness and diversity of the bacterial microbiome in human u-13-14-15-15wedid not observe such trends in mice models. This difference may be related to that it is difficult to re-establish the diversity of the microbiota in laboratory mice due to highly stereotyped rearing conditions in specific pathogen-free facilities. Further work using rewilded mice could provide valuable insights.23Prevotellaceae is known to dominate the gut microbiome in rural populations with a preindustrial lifestyle while a decreased prevalence of Prevotella spp. in Westernized populations is compensated by Bacteroides spp.24Amoebae therefore carry the expected effect of reversing the impact of Western diet on the intestinal microbiome. Indeed, a Pre vote lla-r\ch gut microbiome improves weight loss, cholesterol levels and glucose metabolism.25,26These beneficial effects are likely due to the potential of Prevotella spp. to degrade complex polysaccharides and to produce short chain fatty acids, small molecules well-known for their beneficial impact on host health.

[0071] A bloom of Desulfovibrio spp. has been observed in HFD-fed animals suggesting its association with Westemalized diets.27Indeed, several studies have reported a positive correlation between Desulfovibrio spp. and MS phenotypes including hypercholesterolemia, obesity, type 2 diabetes, artery coronary disease in type 2 diabetes. Conversely, the abundance of Desulfovibrio spp. in type 2 diabetes models was reversed by different beneficial compounds derived from plants (for review see27). Cecal Desulfovibrio abundance was also associated with obesity, dyslipidemia and insulin resistance in pigs fed with a Western-style diet.28

[0072] Here, we observed a general trend toward improvement of the MS in HDF-fed animals colonized by E. muris, but the impact of amoebas was only significant on the hepatic consequences of HFD. In humans, data about the relationship between Prevotellaceae and MAFLD are controversial (for a review, see29). Four studies reported a negative association between MAFLD and Prevotellaceae30313233in agreement with our results. Two studies reported an inverse association between MAFLD and Prevotellaceae .34,35To explain these discrepancies, it is to note that Zhu's work showed no difference between patients and non- obese controls, suggesting that Prevotellaceae were associated with overweight rather than with liver disease. Furthermore, Mickail's work focusing on a small group of children, raised the possibility of age-related differences. Finally, ten studies showed no association between MAFLD and Prevotellaceae ,29

[0073] To our knowledge, no study has reported a significant link between Desulfovibrio spp. and MAFLD in Human.29However, many studies have reported a reduction in their abundance in association with an amelioration of HFD-induced MAFLD in rodents under several nutritional supplements or plant extracts.27Conversely, gavage of HFD-fed mice with Desulfovibrio piger increased hepatic steatosis and fibrosis suggesting a causative role of this bacterium.36 Little is known about the interactions able to explain the relationship between protozoa and bacteria in the intestine. Amoebae have a direct predatory effect on commensal bacteria. Less predation on species of the Prevotella genus and more predation on species of the Desulfovibrio genus could explain the observed changes. The direct effect of bacterial predation could also explain the maintenance of greater microbiome biodiversity.37Nonetheless, we cannot dismiss the possibility that amoebae generate or metabolize chemicals components in the digestive tract that may affect bacterial growth.

[0074] BAs are synthesized from cholesterol in liver and then secreted into the gut. Most BA (90%- 95%) are reabsorbed by intestine and liver while only a small portion (5%— 10%) are excreted into feces.38Gut microbiota converts conjugated BAs into unconjugated secondary BAs which modulate inflammation and tumorigenesis in the intestine, mesenteric fat, and liver.18Deconjugation is mediated via bile salt hydrolases. Neither Prevotella spp. nor Desulfovibrio spp. are known to be key deconjugation actors39suggesting that the decreased deconjugation activity is related to a decrease abundance of alternative bacteria like Bacteroides, which are negatively correlated with Prevotella abundance.

[0075] HFD mice colonized by E. muris are characterized by a lower concentration of FXR agonist BAs. FXR is a key receptor allowing BA signaling in several tissues. It is mainly expressed in the liver and in the intestine where it plays a key role for glucose and lipid control (for review see ref40). Under HFD, FXR deficient mice are usually protected against obesity and exhibit improved glucose homeostasis compared with control mice.41Hepatic deletion of FXR contributes to lipid accumulation in liver via a de novo lipogenesis.42The reduced levels of FXR agonists may thus explain the effect of E. muris on hepatic steatosis.

[0076] In accordance with our data, hepatic steatosis appears as the consequence of hepatic de novo lipogenesis and increased triglyceride storage.43Lipid accumulation results in lipotoxicity and inflammation via activation of Kuppfer cells and macrophages. The activation could be related to the formation of cholesterol crystals with a subsequent activation of the NOD-like receptor protein 3 (NLRP3) / IL10 inflammasome pathway.44However, diverse stimuli including mitochondrial damage, endoplasmic reticulum stress and others may also activate the NLRP3 inflammasome and IL 10 secretion.45 We finally found that the relative abundance of E. muris was correlated with cAMP level in the cecum of HFD-fed mice. cAMP is known to play a major role in the social organization of telluric amoebas (especially Dictyostelium discoideum). Indeed, self-organization of amoebas into aggregates, migrating slugs and fruiting structures is under the control of cAMP signalling.19cAMP in Entamoeba species is less studied but A. histolitica has a cAMP signaling system suggesting that this second messenger also plays a role in commensal amoebaes.46The correlation reported in the present study further points to this direction. cAMP has been shown to improve the impact of HFD on MS in mice. Wang et al. reported that cAMP causes a reduction of the adipose tissue, a decreased size of adipocytes, decreased triglycerides and cholesterol levels in the serum and finally less hepatic steatosis in high-fat fed mice.20The beneficial impact of amoebas in high-fat fed mice may thus also be directly related to cAMP production by amoeba.

[0077] Altogether, E. muris induces a coherent set of changes in HFD-fed mice, all of which are expected to promote protection against MS. In this model, the effect on MS mainly relates to liver lesions, but common protozoa of the Western world (mainly Dientamoeba fragilis and Blastocystis hominis were very recently associated with a lower prevalence of MS in an obese population.47Altogether, these results suggest that the loss of protozoa could be a risk factor associated with modern Western lifestyle that may contribute to the high incidence of MS worldwide. Our finding suggests that the recolonization of our digestive tract by commensal amoebas could be alternative approaches in treating MAFLD.

[0078] REFERENCES:

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Claims

CLAIMS:

1. A method of treating metabolic syndrome in a subject in need thereof comprising administering a therapeutically effective amount of amoebae.

2. The method of claim 1 wherein the subject is overweight.

3. The method of claim 1 wherein the subject is obese or is at risk of obesity.

4. The method according to any of claims 1 to 3 for the treatment of metabolic-associated fatty liver disease (MAFLD).

5. The method according to any of claims 1 to 3 for reducing hepatic steatosis.

6. The method according to any one of claims 1 to 5 wherein the amoeba is not a parasitic amoeba.

7. The method of claim 6 wherein the amoeba is a commensal amoeba.

8. The method of claim 7 wherein the amoeba of is Entamoeba coli.

9. The method according to any one of claims 1 to 8 wherein the amoeba is administered via oral ingestion.

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