Claire Cherbuy

Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.

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.

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.

Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-Butyrate, glucose, and glutamine

BACKGROUND & AIMS Among substrates available to the colonic mucosa, n-butyrate from bacterial origin represents a major fuel. The present work investigated possible modifications of energy substrate metabolism in colonocytes isolated from germfree rats. METHODS Colonocytes isolated from germfree vs. conventional rats were incubated (30 minutes at 37 degrees C) in the presence of 14C-labeled n-butyrate (10 mmol/L), glucose (5 mmol/L), or glutamine (5 mmol/L). 14CO2 and metabolites generated were measured. Possible regulatory steps were also investigated. RESULTS Glucose use rate was 25% lower in germfree rat colonocytes due to a reduced glycolytic capacity in these cells. Differences in 6-phosphofructo-1-kinase activity could account for this decrease. In contrast, glutamine use rate was 45% higher, and this was correlated with a higher maximum velocity of glutaminase in these cells. Nevertheless, the capacities to oxidize glucose and glutamine remained unchanged. Although the capacity to use n-butyrate was maintained in colonocytes of germfree rats, the ketogenic capacity was lower, whereas the capacity to oxidize n-butyrate was higher. The mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein was identified in the colonic mucosa. Moreover, the messenger RNA and amount of protein were 75% lower in the germfree state. CONCLUSIONS The absence of an intestinal microflora induces specific changes in the metabolic capacities of colonocytes.

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

Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat

Previous studies have suggested that intestinal microbiota modulates colonic epithelium renewal. The objective of our work was to study the effects of microbiota on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic crypts and amount of cell cycle-related proteins were compared between germ-free (GF), conventional (CV), and conventionalized rats by histochemistry and Western blot. Ki67 and proliferating cell nuclear antigen (PCNA) were used as surrogates for proliferative cells; p21(cip1) and p27(kip1) were markers of cell cycle arrest; anti- and proapoptotic proteins, Bcl2 and Bax, respectively, were also studied. We observed 40% increase of the crypt proliferative area 2 days after inoculation of GF rats with a complex microbiota. This recruitment of proliferative cells may account for the 30% increase of crypt depth observed between CV and GF rats. The hyperproliferative boost induced by microbiota was compensated by a fourfold increase of p21(cip1) and p27(kip1) involved in cell cycle arrest and a 30% drop of antiapoptotic Bcl2 protein while Bax was unchanged. Inductions of p21(cip1), p27(kip1), and PCNA protein were not paralleled by an increase of the corresponding mRNA. We also showed that p21(cip1) induction by microbiota was partially restored by Bacteroides thetaiotaomicron, Ruminococcus gnavus, and Clostridium paraputrificum. Colonization of the colon by a complex microbiota increases the crypt depth of colon epithelium. This event takes place in conjunction with a multistep process: a hyperproliferative boost accompanied by compensatory events as induction of p21(cip1) and p27(kip1) and decrease of Bcl2.

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.

Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by NH4+

Short chain fatty acids (SCFA) from bacterial origin, as well as glucose from vascular origin, are among fuel substrates available to the colonic mucosa. The present work investigated the possible modulation by another bacterial metabolite, i.e. ammonia, of the capacities of colonic epithelial cells to metabolize these substrates. Viable colonocytes were isolated from the proximal colon of 40–50 kg pigs fed a standard diet and were incubated (30 min, 37°C) in the presence of a concentration range of 14C-labeled n-butyrate or acetate, or 14C-labeled glucose (5 mm), with or without NH4Cl (10 mM) addition. 14CO2 and metabolites generated were measured. Butyrate utilization resulted in a high generation of ketone bodies (acetoacetate and β-OH-butyrate), in addition to 14CO2 production. However, the net ketone body generation was significantly decreased for butyrate concentrations higher than 10 mM. In contrast to n-butyrate, acetate when given as the sole substrate got preferentially metabolized in the oxidation pathway. Acetate metabolism was not affected by NH4Cl, thus indicating that the tricarboxylic acid cycle was unchanged. Conversely, 4C02 and ketone body production from butyrate were decreased by 30% in the presence of NH4Cl, suggesting that butyrate activation or β-oxidation was diminished. Glucose utilization rate was increased by 20%, due to an increased glycolytic capacity in the presence of NH4Cl. A dose-dependent stimulation of phosphofructokinase activity by NH4+ could account for this effect. It is concluded that ammonia, whose physiological concentration is high in the colonic lumen, can modulate the metabolism of two major substrates, n-butyrate and glucose, in colonic epithelial cells.

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.

Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cows milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.