Marie-Louise Noordine

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.

Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome

In short bowel syndrome (SBS), although a remaining colon improves patient outcome, there is no direct evidence of a mucosal colonic adaptation in humans. This prospective study evaluates morphology, proliferation status, and transporter expression level in the epithelium of the remaining colon of adult patients compared with controls. The targeted transporters were Na+/H+ exchangers (NHE2 and 3) and oligopeptide transporter (PepT1). Twelve adult patients with a jejuno-colonic anastomosis were studied at least 2 yr after the last surgery and compared with 11 healthy controls. The depth of crypts and number of epithelial cells per crypt were quantified. The proliferating and apoptotic cell contents were evaluated by revealing Ki67, PCNA, and caspase-3. NHE2, NHE3, PepT1 mRNAs, and PepT1 protein were quantified by quantitative RT-PCR and Western blot, respectively. In patients with SBS compared with controls, 1) hyperphagia and severe malabsorption were documented, 2) crypt depth and number of cells per crypt were 35% and 22% higher, respectively (P < 0.005), whereas the proliferation and apoptotic levels per crypt were unchanged, and 3) NHE2 mRNA was unmodified; NHE3 mRNA was downregulated near the anastomosis and unmodified distally, and PepT1 mRNA and protein were unmodified. We concluded that, in hyperphagic patients with SBS with severe malabsorption, adaptive colonic changes include an increased absorptive surface with an unchanged proliferative/apoptotic ratio and well-preserved absorptive NHE2, NHE3, and PepT1 transporters. This is the first study showing a controlled nonpharmacological hyperplasia in the colon of patients with SBS.

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.

Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats

The thermophilic lactic acid bacterium Streptococcus thermophilus is widely and traditionally used in the dairy industry. Despite the vast level of consumption of S. thermophilus through yogurt or probiotic functional food, very few data are available about its physiology in the gastrointestinal tract (GIT). The objective of the present work was to explore both the metabolic activity and host response of S. thermophilus in vivo. Our study profiles the protein expression of S. thermophilus after its adaptation to the GIT of gnotobiotic rats and describes the impact of S. thermophilus colonization on the colonic epithelium. S. thermophilus colonized progressively the GIT of germ-free rats to reach a stable population in 30 days (108 cfu/g of feces). This progressive colonization suggested that S. thermophilus undergoes an adaptation process within GIT. Indeed, we showed that the main response of S. thermophilus in the rats GIT was the massive induction of the glycolysis pathway, leading to formation of lactate in the cecum. At the level of the colonic epithelium, the abundance of monocarboxylic acid transporter mRNAs (SLC16A1 and SLC5A8) and a protein involved in the cell cycle arrest (p27kip1) increased in the presence of S. thermophilus compared with germ-free rats. Based on different mono-associated rats harboring two different strains of S. thermophilus (LMD-9 or LMG18311) or weak lactate-producing commensal bacteria (Bacteroides thetaiotaomicron and Ruminococcus gnavus), we propose that lactate could be a signal produced by S. thermophilus and modulating the colon epithelium.

The microbiota shifts the iron sensing of intestinal cells

The amount of iron in the diet directly influences the composition of the microbiota. Inversely, the effects of the microbiota on iron homeostasis have been little studied. So, we investigate whether the microbiota itself may alter host iron sensing. Duodenal cytochrome b and divalent metal transporter 1, involved in apical iron uptake, are 8‐ and 10‐fold, respectively, more abundant in the duodenum of germ‐free (GF) mice than in mice colonized with a microbiota. In contrast, the luminal exporter ferroportin is 2‐fold less abundant in GF. The overall signature of microbiota on iron‐related proteins is similar in the colon. The colonization does not modify systemic parameters as plasma transferrin saturation (20%), plasma ferritin (150 ng/L), and liver (85 μg/g) iron load. Commensal organisms (Bacteroides thetaiotaomicron VPI‐5482 and Faecalibacterium prausnitzii A2‐165) and a probiotic strain (Streptococcus thermophilus LMD‐9) led to up to 12‐fold induction of ferritin in colon. Our data suggest that the intestinal cells of GF mice are depleted of iron and that following colonization, the epithelial cells favor iron storage. This study is the first to demonstrate that gut microbes induce a specific iron‐related protein signature, highlighting new aspects of the crosstalk between the microbiota and the intestinal epithelium.—Deschemin, J.‐C., Noordine, M.‐L., Remot, A., Willemetz, A., Afif, C., Canonne‐Hergaux, F., Langella, P., Karim, Z., Vaulont, S., Thomas, M., Nicolas, G. The microbiota shifts the iron sensing of intestinal cells. FASEB J. 30, 252‐261 (2016). www.fasebj.org

Bacteria isolated from lung modulate asthma susceptibility in mice.

Asthma is a chronic, non-curable, multifactorial disease with increasing incidence in industrial countries. This study evaluates the direct contribution of lung microbial components in allergic asthma in mice. Germ-Free and Specific-Pathogen-Free mice display similar susceptibilities to House Dust Mice-induced allergic asthma, indicating that the absence of bacteria confers no protection or increased risk to aeroallergens. In early life, allergic asthma changes the pattern of lung microbiota, and lung bacteria reciprocally modulate aeroallergen responsiveness. Primo-colonizing cultivable strains were screened for their immunoregulatory properties following their isolation from neonatal lungs. Intranasal inoculation of lung bacteria influenced the outcome of allergic asthma development: the strain CNCM I 4970 exacerbated some asthma features whereas the pro-Th1 strain CNCM I 4969 had protective effects. Thus, we confirm that appropriate bacterial lung stimuli during early life are critical for susceptibility to allergic asthma in young adults.

264 Bacteroides Thetaiotaomicron and Faecalibacterium prausnitzii Shape the Mucus Production and Mucin O-Glycosylation in Colon Epithelium

The intestinal mucus layer plays a key role in themaintenance of host-microbiota homeostasis. The production of goblet cells, which secrete mucus, is modulated by microbiota, but neither the species nor the mechanisms involved in this process are still unknown. We studied how two prominent commensal bacteria may influence the mucus production by goblet cells and the profile of mucin glycosylation in gnotobiotic rats. We have chosen Bacteroides thetaiotaomicron, which is characterized by its high mucus-polysaccharides degrading potential, and Faecalibacterium prausnitzii, which is a sensor of intestinal health. Germ free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii leading respectively to mono-associated (Bt-rats) and di-associated rats (Bt+Fp-rats). A panel of goblet cells markers was analyzed by histological staining, immunohistochemistry, quantitative PCR and Western blot in colon epithelium. The mucin O-glycosylation was determined by MALDI TOF mass spectrometry. In Bt-rats, the goblet cells number and the expression of mucus-related genes (muc2, muc4, klf4, c1galt1 and b4galt4 mRNAs) were increased compared to GF ones. KLF4 protein, a transcription factor involved in goblet cell terminal differentiation, was also increased in Bt-rats, whereas a decrease in Chromogranin A protein, a marker of enteroendocrine cells was observed. We propose that B. thetaiotaomicron provokes an imbalance inside the secretory lineage by favoring mucus production at the expense of enteroendocrine cells. When B. thetaiotaomicron was associated to F. prausnitzii, the effects on goblet cells were reduced/ decreased/diminished. Indeed, the number of goblet cells per crypt and the amount of KLF4 protein were lower in Bt+Fp-rats than in Bt-rats. We then analyzed the mucus quality by studying the profile of mucin O-glycosylation. In Bt-rats, a decrease in the production of sulfated (4.5% of total oligosaccharides instead of 12.9%) and neutral (40.1% instead of 52.8%) oligosaccharides was observed and was correlated to an increased proportion of sialylated O-glycans carrying NeuAc (24.2% instead of 18.9%) or NeuGc (31.2% instead of 15.4%) residues compared to GF rats. Thus, B. thetaiotaomicron impacts the composition of mucin O-glycans, with a decrease in sulfated and neutral oligosaccharides in favor of sialylated ones. Furthermore, glycosylation of mucins from Bt+Fp-rats resembled to those of GF rats. As previously observed for goblet cells, F. prausnitzii seemed to decrease the effect of B. thetaiotaomicron on mucus. Using a novel gnotobiotic model, which is the first described with F. prausnitzii, we showed how the balance between B. thetaiotaomicron and F. prausnitzii plays a key role in protecting epithelium via their respective effects on mucus.

1058 Crosstalk Between Commensal Bacteria and Colonic Epithelium in Gnotobiotic Rats: A Key Role of Acetate

The gastrointestinal tract (GIT) of mammals is colonized by a community of micro-organisms reaching levels as high as 1011 bacteria/g of contents in the colon. This microbiota is involved in intestinal homeostasis and epithelium structure through the control of proliferation and differentiation processes (Cherbuy et al., 2010). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, dominant members of microbiota, are commensal model bacteria to study host-microbiota interactions. The objective of our work was to understand the effects of these two bacteria on colonic epithelium homeostasis in gnotobiotic rats and underlying this crosstalk using In Vitro models. Germ-free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii. Colonic epithelium morphology, crypt depth, cell cycle-related proteins and goblet cells markers were analyzed by histological staining, immunohistochemistry and Western blot in GF (n= 6), mono-associated (Ino-Bt; n=12) or di-associated (Ino-Bt+Fp; n=6) rats 30 days after inoculation. In Ino-Bt rats, no effect was observed neither on crypt depth nor on proliferation markers whereas Kruppel Like Factor 4 protein (KLF4), a transcription factor involved in goblet cells differentiation, was significantly increased (2.5 ±0.8-fold) compared to GF rats. In parallel with this KLF4 induction, a significant increase of goblet cells per crypt in the colon of Ino-Bt compared to GF rats was observed. In Ino-Bt+Fp rats, there was no induction of KLF4 protein, suggesting that the presence of F. prausnitzii impaired the mucus-stimulatory effect triggered by B. thetaiotaomicron. Short Chain Fatty Acids (SCFA) composition was determined on caecal contents by gas chromatograph to assess bacterial metabolic activity. In Ino-Bt rats, acetate was the major SCFA detected (5.8 μmol/g) with a low level of propionate (1.0 μmol/g) and no butyrate. In Ino-Bt+Fp rats, butyrate was the major SCFA (1.3 μmol/g) and acetate concentration was significantly decreased (two-fold) compared to Ino-Bt rats (3.0 μmol/g). Using HT29-MTX cells, a cell line producing mucus, we showed that acetate was able to increase significantly KLF4 protein whereas butyrate and propionate have no effect on KLF4 level. All of these results suggested that acetate, a bacterial metabolite produced by B. thetaiotaomicron, could mediate a bacterial signal to promote goblet cells differentiation pathway in the GIT of mono-associated rats. In Crohns disease patients, a diminished prevalence of F. prausnitzii (Martinez-Medina et al., 2006) and an increase of mucus production (Gersemann et al., 2009) have been often reported. Our observations in gnotobiotic rats are coherent with reports made in patients and may provide mechanistic clues to understand the pathophysiology of inflammatory bowel diseases.