These integrin-mediated interactions, on either side of the plasma membrane, are dynamically linked. The cytoskeleton controls the functional state of the integrins thus modulating their interaction with the ECM. Meanwhile integrin binding to the ECM changes the cell shape and the composition of the cytoskeleton beneath [8]. The nonpathogenic yeast Saccharomyces boulardii (Sb) is widely used in a lyophilized form to treat and prevent antibiotic- associated and infectious diarrhea [9]. Recent in vitro and in vivo studies indicate that Sb interacts not only with pathogenic micro- organisms and resident microflora, but also with intestinal mucosa [10,11]. In addition Sb has been shown to exert a trophic effect that restores intestinal homeostasis [11]. Furthermore, clinical trials have suggested that Sb can be effective in the treatment of IBD [12,13] through the modulation of host cell signaling pathways implicated in the proinflammatory response [14,15, 16]. However, no information is currently available on the possible effects of Sb upon colonic epithelial cell restitution. In the present study, we show that Sb secretes factors that modulate intestinal epithelial cell restitution both in vitro and in vivo. Our findings suggest that Sb increases intestinal epithelial cell migration without affecting cell proliferation. Sb exerts at least some of its motogenic effects through the activation of the a2b1 integrin collagen receptor signaling pathway, which is associated with the reorganization of focal adhesions. Methods Cell culture The human colonic adenocarcinoma cell lines HCT-8/E11, CaCo2/TC7, HT29-D4 and T84, were routinely cultured as previously described [17,18,19,20]. Cells were cultured on plastic dishes until they reached confluency. These cellular monolayers consist of polarized cells joined by tight junctions, exhibiting well developed apical microvilli, which allow the study of the processes involved in intestinal epithelial cell physiology. Yeast culture supernatants The supernatants of Sb (Biocodex laboratories; Gentilly, France) and Saccharomyces cerevisiae (Sc, strain BY4742, a gift from C. De La Roche Saint-Andre´; CNRS, Marseille, France) were prepared as previously described [21]. Briefly, yeast strains (100 mg/ml) were cultured overnight at 37uC in epithelial cell culture media. Conditioned media were centrifuged at 20,0006 g for 15 minutes and the supernatants collected. The supernatants were passed through 0.22 mm filters (Fisher Scientific) to remove yeast cells. Serial dilutions ranging from 1/8 to 1/128 were performed in epithelial cell culture media. None of the diluted supernatants affected cell viability, as verified by the trypan blue exclusion test. Wound healing model Monolayers of differentiated cells were wounded using a sterile tooth-pick and incubated with or without various dilutions of Sb supernatant. Plates were placed in a temperature and CO2- controlled chamber mounted on a Nikon TE2000 inverted microscope. Images were captured every 5 minutes for a total observation period of 5 hours using a Cool SnapHQ camera (Princeton Instruments) through a 106 objective lens. For each wound, 10 measurements of wound width were recorded. To assess the role of the integrins in Sb enhanced enterocyte migration, 10 mg/ml function-blocking anti-integrin monoclonal antibodies (mAbs) (GoH3 (anti-a6), 69.6.5 (anti-av), Lia1/2 (anti-b1), Gi9 (anti-a2), and SAM-1 (anti-a5); Beckman Coulter, Marseille, France), b1-activating mAb (TS2/16; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti DPP IV mAb (S. Maroux, CNRS, S. boulardii Enhances Cell Restitution Marseille) were added 1 hour before wounding and again during the period of cell migration. In some experiments, MAPK inhibitor (10 mM PD 98059; Tocris Bioscience, Bristol, UK ) or 10 nM FAK inhibitor (PF 573228; Tocris Bioscience) were added 1 hour before wounding and again during the period of cell migration. When required, cells were treated with 2.5 mg/mL mitomycin C (Sigma) prior and during cell migration in order to block cell proliferation. In order to track the rate of cell proliferation, 10 mmol/L 5-bromo- 29-deoxyuridine (BrdU) was added 4 hours after the wound and incubation continued for an additional 1 hour period. Cells that had incorporated BrdU into their DNA were detected using the BrdU labeling and detection kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). Cell proliferation in wounded monolayers was visualized by light microscopy using an inverted Olympus CKX41 microscope. Measurement of enterocyte crypt-villus migration Groups of 6-week old C57BL6J female mice (n = 5) were force-fed daily with 200 mL PBS solution with or without 1 mg/ml lyophilized Sb for 1 week. When required, mice were force-fed daily for one week with 200 mL Sb supernatant or Sc supernatant. Animals were injected with BrdU (50 mg/kg, BrdU; Sigma) intraperitoneally and then sacrificed 24 hours later. Segments of intestine were frozen in liquid nitrogen and cryosectioning (section thickness: 8 mm) was performed. Samples were immunostained with a rat anti-BrdU antibody mAb (Abcam) overnight at 4uC. After washing, sections were incubated with AlexaFluor 488-conjugated goat anti-rat IgG (Invitrogen) for 1 hour, and then mounted in ProLong Gold medium containing DAPI (Invitrogen) in order to couterstain nuclei. Images were captured and analyzed using a Leica DM IRBE microscope. Enterocyte migration was determined by measuring the distance from the bottom of the crypt to the foremost labeled enterocyte and expressing the distance as a percentage of the total villus height. All animal experiments were performed in accordance with the regulations of our institution’s ethics commission. They were conducted following the APS Guiding Principles in the Care and Use of Animals. The study was approved by the Ethics Committee in Animal Experimentation of Centre Me´diterrane´en de Me´decine Mole´culaire (C3M), Nice, France (protocol 3/2010). Cell adhesion assay Cell adhesion assays on Laminin-111 (Sigma, Saint Quentin Fallavier, France) and type I collagen (BD-Bioscience, Belford, MA, USA) were performed using cells incubated with or without Sb supernatant, as previously published [22]. Function blocking anti-integrin mAbs (10 mg/mL) were added to a subset of samples, to identify the receptor types involved in Sb enhanced adhesion. Immunocytochemistry and Immunohistochemistry Cells, incubated with or without Sb supernatant, were fixed and permeabilized as previously described [23]. Paxillin was immuno- stained with a mouse anti-paxillin mAb (Millipore). Tissues were fixed in acetone for 10 minutes at -20uC, and stained with rat anti- ki-67 (Abcam), rat anti-a6 integrin (Beckman coulter), rabbit anti- a2 integrin (Millipore) or rabbit anti-Y118 Paxillin (Millipore) Abs. After washing, samples were incubated with the appropriate Alexa 488- or Alexa 546-conjugated secondary Abs (Invitrogen), and then mounted in ProLong Gold medium. Images were captured and analyzed using a Leica DM IRBE microscope. Detection of tyrosine-phosphorylated proteins Single-cell suspensions (25 000 cells/0.1 mL), prepared in DMEM containing 0.2% BSA (adhesion buffer), were seeded onto PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e18427