title: Multispecies probiotic protects gut barrier function in experimental models.
abstract: OBJECTIVE
  To investigate the effect of the probiotic combination Lactibiane Tolerance(®) (LT) on epithelial barrier function in vitro and in vivo.
  
  
  METHODS
  The effect of the multispecies probiotic LT was assessed on several models of epithelial barrier function both in vitro (in basal and inflammatory conditions) and in vivo [visceral hypersensitivity induced by chronic stress or by colonic perfusion of a fecal supernatant (FSN) from patients with irritable bowel syndrome (IBS)]. In vitro, we measured the permeability of confluent T84 cell monolayers incubated with or without LT by evaluating the paracellular flux of macromolecules, in basal conditions and after stimulation with lipopolysaccharide (LPS) or with conditioned medium of colonic biopsies from IBS patients (IBS-CM). In vivo, male C57/Bl6 mice received orally NaCl or LT for 15 d and were submitted to water avoidance stress (WAS) before evaluating visceral sensitivity by measuring the myoelectrical activity of the abdominal muscle and the paracellular permeability with (51)Cr-EDTA. Permeability and sensitivity were also measured after colonic instillation of FSN. Tight-junctions were assessed by immunoblotting and TLR-4 expression was evaluated by immunohistochemistry
  
  
  RESULTS
  Incubation of T84 cell monolayers with LT in basal conditions had no significant effect on permeability (P > 0.05 vs culture medium). By contrast, addition of LT bacterial bodies (LT) completely prevented the LPS-induced increase in paracellular permeability (P < 0.01 vs LPS 10 ng/mL (LPS 10); P < 0.01 vs LPS 100 ng/mL (LPS 100), P > 0.05 vs culture medium). The effect was dose dependent as addition of 10(9) LT bacterial bodies induced a stronger decrease in absorbance than 10(6) LT (10(9) LT + LPS 10: -20.1% ± 13.4, P < 0.01 vs LPS 10; 10(6) LT + LPS 10: -11.6% ± 6.2, P < 0.01 vs LPS 10; 10(9) LT + LPS 100: -14.4% ± 5.5, P < 0.01 vs LPS 100; 10(6) LT + LPS 100: -11.6% ± 7.3, P < 0.05 vs LPS 100). Moreover, the increase in paracellular permeability induced by culturing T84 cells with conditioned medium of colonic biopsies from IBS patients (IBS-CM) was completely inhibited in the presence of 10(9) LT (P < 0.01 vs IBS-CM). LT also significantly prevented the epithelial disruption induced by intracolonic infusion of fecal supernatant from IBS patients (P < 0.01 vs IBS FSN) or water avoidance stress P < 0.01 vs WAS) in C57/Bl6 mice and increased the expression of occludin in vitro and in vivo, as assessed by immnunoblotting. The WAS-induced effect on visceral sensitivity was prevented by LT treatment since values obtained for all steps of colorectal distension were significantly (P < 0.01) different from the WAS group. Finally, LT down-regulated the response mediated through TLR-4 in vitro (decrease in tumor necrosis factor α secretion in response to LPS: -65.8% for 10(9) LT and -52.5% for 10(6) LT, P < 0.01 vs LPS) and in vivo (inhibition of WAS induced an increase in TLR-4 expression in the LT treated mice colon, P < 0.01 vs WAS).
  
  
  CONCLUSIONS
  The probiotic LT mix prevented the disruption to the epithelial barrier induced by LPS, stress or colonic soluble factors from IBS patients and prevented visceral hypersensitivity.
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Mylene
authors.initials: M
authors.lastname: Nébot-Vivinus
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Cherryl
authors.initials: C
authors.lastname: Harkat
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Hanene
authors.initials: H
authors.lastname: Bzioueche
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Christel
authors.initials: C
authors.lastname: Cartier
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Plichon-Dainese
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Lara
authors.initials: L
authors.lastname: Moussa
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Helene
authors.initials: H
authors.lastname: Eutamene
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Dorsa
authors.initials: D
authors.lastname: Pishvaie
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Holowacz
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Seyrig
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Piche
authors.affiliation: Mylene Nébot-Vivinus, Hanene Bzioueche, Department of Immunology, Pole of Biology, CHU de Nice, 06103 Nice, France.
authors.forename: Vassilia
authors.initials: V
authors.lastname: Theodorou
mesh_terms: D000818:Animals; D002460:Cell Line; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D006801:Humans; D006930:Hyperalgesia / Q000378:metabolism / Q000382:microbiology / Q000517:prevention & control; D018836:Inflammation Mediators / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D043183:Irritable Bowel Syndrome / Q000378:metabolism / Q000382:microbiology / Q000628:therapy*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008810:Mice, Inbred C57BL; D010539:Permeability; D019936:Probiotics / Q000627:therapeutic use*; D062725:Tight Junction Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism / Q000382:microbiology; D013997:Time Factors; D046509:Tissue Culture Techniques; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D059265:Visceral Pain / Q000378:metabolism / Q000382:microbiology / Q000517:prevention & control
chemical_list: D017077:Culture Media, Conditioned; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D062725:Tight Junction Proteins; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha
keywords: Hypersensitivity; Intestinal epithelial barrier permeability; Irritable bowel syndrome; Probiotic