Muriel Thomas

Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent

BackgroundThe intestinal mucus layer plays a key role in the maintenance of host-microbiota homeostasis. To document the crosstalk between the host and microbiota, we used gnotobiotic models to study the influence of two major commensal bacteria, Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, on this intestinal mucus layer. B. thetaiotaomicron is known to use polysaccharides from mucus, but its effect on goblet cells has not been addressed so far. F. prausnitzii is of particular physiological importance because it can be considered as a sensor and a marker of human health. We determined whether B. thetaiotaomicron affected goblet cell differentiation, mucin synthesis and glycosylation in the colonic epithelium. We then investigated how F. prausnitzii influenced the colonic epithelial responses to B. thetaiotaomicron.ResultsB. thetaiotaomicron, an acetate producer, increased goblet cell differentiation, expression of mucus-related genes and the ratio of sialylated to sulfated mucins in mono-associated rats. B. thetaiotaomicron, therefore, stimulates the secretory lineage, favoring mucus production. When B. thetaiotaomicron was associated with F. prausnitzii, an acetate consumer and a butyrate producer, the effects on goblet cells and mucin glycosylation were diminished. F. prausnitzii, by attenuating the effects of B. thetaiotaomicron on mucus, may help the epithelium to maintain appropriate proportions of different cell types of the secretory lineage. Using a mucus-producing cell line, we showed that acetate up-regulated KLF4, a transcription factor involved in goblet cell differentiation.ConclusionsB. thetaiotaomicron and F. prausnitzii, which are metabolically complementary, modulate, in vivo, the intestinal mucus barrier by modifying goblet cells and mucin glycosylation. Our study reveals the importance of the balance between two main commensal bacteria in maintaining colonic epithelial homeostasis via their respective effects on mucus.

Identification of Metabolic Signatures Linked to Anti-Inflammatory Effects of Faecalibacterium prausnitzii

ABSTRACT Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable and stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood. IMPORTANCE Inflammatory bowel diseases (IBD) are characterized by low proportions of F. prausnitzii in the gut microbiome. This commensal bacterium exhibits anti-inflammatory effects through still unknown mechanisms. Stable monoassociated rodents are actually not a reproducible model to decipher F. prausnitzii protective effects. We propose a new gnotobiotic rodent model providing mechanistic clues. In this model, F. prausnitzii exhibits protective effects against an acute colitis and a protective metabolic profile is linked to its presence along the digestive tract. We identified a molecule, salicylic acid, directly involved in the protective effect of F. prausnitzii. Targeting its metabolic pathways could be an attractive therapeutic strategy in IBD. Inflammatory bowel diseases (IBD) are characterized by low proportions of F. prausnitzii in the gut microbiome. This commensal bacterium exhibits anti-inflammatory effects through still unknown mechanisms. Stable monoassociated rodents are actually not a reproducible model to decipher F. prausnitzii protective effects. We propose a new gnotobiotic rodent model providing mechanistic clues. In this model, F. prausnitzii exhibits protective effects against an acute colitis and a protective metabolic profile is linked to its presence along the digestive tract. We identified a molecule, salicylic acid, directly involved in the protective effect of F. prausnitzii. Targeting its metabolic pathways could be an attractive therapeutic strategy in IBD.

Drastic changes in fecal and mucosa-associated microbiota in adult patients with short bowel syndrome

Short bowel syndrome (SBS) is observed in Humans after a large resection of gut. Since the remnant colon and its associated microbiota play a major role in the outcome of patients with SBS, we studied the overall qualitative and quantitative microbiota composition of SBS adult patients compared to controls. The population was composed of 11 SBS type II patients (with a jejuno-colonic anastomosis) and 8 controls without intestinal pathology. SBS patients had 38 +/- 30 cm remnant small bowel length and 66 +/- 19% of residual colon. The repartition of proteins, lipids, carbohydrates and fibres was expressed as % of total oral intake in patients and controls. The microbiota was profiled from stool and biopsy samples with temporal temperature gradient gel electrophoresis and quantitative PCR. We show here that microbiota of SBS patients is unbalanced with a high prevalence of Lactobacillus along with a sub-dominant presence and poor diversity of Clostridium leptum, Clostridium coccoides and Bacteroidetes. In addition, Lactobacillus mucosae was detected within the fecal and mucosa-associated microbiota of SBS patients, whereas it remained undetectable in controls. Thus, in SBS the microbial composition was deeply altered in fecal and mucosal samples, with a shift between dominant and sub-dominant microbial groups and the prevalence of L. mucosae.

Lactobacilli as multifaceted probiotics with poorly disclosed molecular mechanisms

Lactic acid bacteria and more particularly lactobacilli have been used for the production of fermented foods for centuries. Several lactobacilli have been recognized as probiotics due to their wide range of health-promoting effects in humans. However, little is known about the molecular mechanisms underpinning their probiotic functions. Here we reviewed the main beneficial effects of lactobacilli and discussed, when the information is available, the molecular machinery involved in their probiotic function. Among the beneficial effects, lactobacilli can improve digestion, absorption and availability of nutrients. As an example, some strains are able to degrade carbohydrates such as lactose or α-galactosides that may cause abdominal pain. Furthermore, they can hydrolyze compounds that limit the bioavailability of minerals like tannin and phytate due to tannin acylhydrolase and phytase activities. In addition, it was shown that some lactobacilli strains can improve mineral absorption in Caco-2 cells. Lactobacilli can also contribute to improve the nutritional status of the host by producing B group vitamins. More recently, the role of lactobacilli in energy homeostasis, particularly in obese patients, is the object of an increased interest. Lactobacilli are also involved in the prevention of diseases. They have potential to prevent carcinogenesis through the modulation of enzymes involved in the xenobiotic pathway, and may prevent cardiovascular diseases such as hypertension through the production of a bioactive peptide that may have angiotensin converting enzyme inhibitor activity. Lactobacilli are increasingly studied for the treatment of inflammatory bowel diseases and exhibit interesting potential in the reduction of pain perception. The ability of some strains to bind to intestinal cells, their pathogen-associated molecular patterns and the metabolites they produce confer interesting immunomodulatory effects. Finally, pathogenic fungi, virus or bacteria can be inhibited by probiotics. They can reinforce the intestinal barrier, simply occupying the ecological niche, or they can have an active role by synthesizing various metabolites inhibiting pathogen development. Lactobacilli have a long standing history with foods and humans but comparatively, their history as probiotics is recent. Their effects are investigated in in vitro and in vivo models leading sometimes to contradictory or controversial results that make necessary final demonstrations through clinical trials. Researches on molecular mechanisms involved in the beneficial effects reviewed here are necessary for a better understanding of these effects, but could also lead to the development of molecular tools to help the screening of the probiotic potential of lactobacilli that are common inhabitants of numerous fermented foods around the world.

Lactobacillaceae and Cell Adhesion: Genomic and Functional Screening

The analysis of collections of lactic acid bacteria (LAB) from traditional fermented plant foods in tropical countries may enable the detection of LAB with interesting properties. Binding capacity is often the main criterion used to investigate the probiotic characteristics of bacteria. In this study, we focused on a collection of 163 Lactobacillaceace comprising 156 bacteria isolated from traditional amylaceous fermented foods and seven strains taken from a collection and used as controls. The collection had a series of analyses to assess binding potential for the selection of new probiotic candidates. The presence/absence of 14 genes involved in binding to the gastrointestinal tract was assessed. This enabled the detection of all the housekeeping genes (ef-Tu, eno, gap, groEl and srtA) in the entire collection, of some of the other genes (apf, cnb, fpbA, mapA, mub) in 86% to 100% of LAB, and of the other genes (cbsA, gtf, msa, slpA) in 0% to 8% of LAB. Most of the bacteria isolated from traditional fermented foods exhibited a genetic profile favorable for their binding to the gastrointestinal tract. We selected 30 strains with different genetic profiles to test their binding ability to non-mucus (HT29) and mucus secreting (HT29-MTX) cell lines as well as their ability to degrade mucus. Assays on both lines revealed high variability in binding properties among the LAB, depending on the cell model used. Finally, we investigated if their binding ability was linked to tighter cross-talk between bacteria and eukaryotic cells by measuring the expression of bacterial genes and of the eukaryotic MUC2 gene. Results showed that wild LAB from tropical amylaceous fermented food had a much higher binding capacity than the two LAB currently known to be probiotics. However their adhesion was not linked to any particular genetic equipment.

Primocolonization is associated with colonic epithelial maturation during conventionalization

Interaction between the gut microbiota and the host starts immediately after birth with the progressive colonization of the sterile intestine. Our aim was to investigate the interactions taking place in the colonic epithelium after the first exposure to gut microbiota. Germ‐free (GF) rats were inoculated with two different microbiotas: the first, obtained from suckling rats, was rich in primocolonizing bacteria and the second, obtained from adult rats, was representative of a mature microbiota. Once transferred into GF rats, these two microbiotas evolved such that they converged, and recapitulated the primocolonization pattern, mimicking the chronological scheme of implantation following birth. The two microbiotas induced common responses in the colonic epithelium: a transitory proliferative phase followed by a compensatory phase characterized by increases in the abundance of p21Cip1 and p27Kip1 and in the number of goblet cells. The effects of the two microbiotas diverged only through their effects on colonic transporters. Analyses of solute carriers and aquaporins revealed that functional maturation was more pronounced following exposure to adult microbiota than suckling microbiota. The colon matured in parallel with the evolution of the microbiota composition, and we therefore suggest a link between intestinal events regulating homeostasis of the colon and modulation of microbial composition.—Tomas, J., Wrzosek, L., Bouznad, N., Bouet, S., Mayeur, C., Noordine, M.‐L., Honvo‐Houeto, E., Langella, P., Thomas, M., Cherbuy, C. Primocolonization is associated with colonic epithelial maturation during conventionalization. FASEB J. 27, 645–655 (2013). www.fasebj.org

Faecal D/L lactate ratio is a metabolic signature of microbiota imbalance in patients with short bowel syndrome.

Our objective was to understand the functional link between the composition of faecal microbiota and the clinical characteristics of adults with short bowel syndrome (SBS). Sixteen patients suffering from type II SBS were included in the study. They displayed a total oral intake of 2661±1005 Kcal/day with superior sugar absorption (83±12%) than protein (42±13%) or fat (39±26%). These patients displayed a marked dysbiosis in faecal microbiota, with a predominance of Lactobacillus/Leuconostoc group, while Clostridium and Bacteroides were under-represented. Each patient exhibited a diverse lactic acid bacteria composition (L. delbrueckii subsp. bulgaricus, L. crispatus, L. gasseri, L. johnsonii, L. reuteri, L. mucosae), displaying specific D and L-lactate production profiles in vitro. Of 16 patients, 9/16 (56%) accumulated lactates in their faecal samples, from 2 to 110 mM of D-lactate and from 2 to 80 mM of L-lactate. The presence of lactates in faeces (56% patients) was used to define the Lactate-accumulator group (LA), while absence of faecal lactates (44% patients) defines the Non lactate-accumulator group (NLA). The LA group had a lower plasma HCO3− concentration (17.1±2.8 mM) than the NLA group (22.8±4.6 mM), indicating that LA and NLA groups are clinically relevant sub–types. Two patients, belonging to the LA group and who particularly accumulated faecal D-lactate, were at risk of D-encephalopathic reactions. Furthermore, all patients of the NLA group and those accumulating preferentially L isoform in the LA group had never developed D-acidosis. The D/L faecal lactate ratio seems to be the most relevant index for a higher D- encephalopathy risk, rather than D- and L-lactate faecal concentrations per se. Testing criteria that take into account HCO3− value, total faecal lactate and the faecal D/L lactate ratio may become useful tools for identifying SBS patients at risk for D-encephalopathy.

Ecology and metabolism of the beneficial intestinal commensal bacterium Faecalibacterium prausnitzii.

Faecalibacterium prausnitzii is a major commensal bacterium, and its prevalence is often decreased in conditions of intestinal dysbiosis. The phylogenic identity of this bacterium was described only recently. It is still poorly characterized, and its specific growth requirements in the human gastrointestinal tract are not known. In this review, we consider F. prausnitzii metabolism, its ecophysiology in both humans and animals, and the effects of drugs and nutrition on its population. We list important questions about this beneficial and ubiquitous commensal bacterium that it would be valuable to answer.

Repetitive Treatments of Colon HT-29 Cells with Diallyl Disulfide Induce a Prolonged Hyperacetylation of Histone H3 K14

Abstract: Diallyl disulfide (DADS) is a sulfur compound derived from garlic. Several studies carried out in rodents have revealed protective effects of DADS against colon carcinogenesis. The antipromoting effects of DADS may be partly related to its ability to inhibit tumoral cell proliferation. In a previous study, we have shown that in two human colon tumor cell lines (HT‐29 and Caco‐2) seeded at a low density (0.2 × 106 cells/100‐mm petri dish), DADS antiproliferative effects were associated with a transient increase of histone H3 K14 acetylation. Moreover, DADS could inhibit nuclear histone deacetylase activity. Therefore, in the present study, we examined the possible effects of different experimental conditions (HT‐29 cells at high density, repetitive treatments with DADS) on the pattern of DADS‐induced histone hyperacetylation. Using HT‐29 cells seeded at a higher density (5 × 106 cells/100‐mm petri dish), we found that DADS induced histone H3 K14 hyperacetylation rapidly (3 h). When administrated as single treatments, the DADS effect on histone H3 K14 remained transient. In contrast, repetitive treatment with DADS resulted in a prolonged hyperacetylation of histone H3 K14. Whatever the cell culture conditions were, DADS had no effect on histone H4 acetylation. Thus, in vitro, the cell density and pattern of DADS treatment influenced the HT‐29 nuclear response to DADS. DADS belongs to food‐borne molecules that may play a role in chromatin remodeling and contribute to the nutritional modulation of gene expression.

Fecal microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice

BACKGROUND & AIMS Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.