mation across the plasma membrane and facilitate interactions between the ECM and the actin cytoskeleton. These integrin- mediated interactions are dynamically linked between either sides of the plasma membrane. 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 [11]. Integrin expression within the intestinal epithelium has been shown to vary, depending on their position along the crypt-villus- axis [12]. This suggests that these molecules are involved in epithelial cell migration. Moreover, during restitution, some integrins undergo a significant level of reorganization [13]. The probiotic yeast Saccharomyces boulardii (Sb) is widely used in a lyophilized form to treat and prevent antibiotic-associated and infectious diarrhea [14]. Recent in vitro and in vivo studies indicate that this probiotic interacts with pathogenic micro-organisms and resident microflora, as well as intestinal mucosa [15,16,17]. In addition, clinical trials have suggested that Sb can be effective in the treatment of inflammatory bowel diseases (IBD) [18,19,20] via modulation of host cell signaling pathways implicated in the pro- inflammatory response [3,21,22]. Furthermore, we have recently shown both in vitro and in vivo that Sb secretes factors that modulate intestinal epithelial cell restitution. This is in part through the activation of the a2b1 integrin collagen receptor signaling pathway [15]. However a2b1 integrin is not the sole cell-ECM receptor involved in colonic restitution, since a3b1 integrin/laminin and avb5 integrin/vitronectin (Vn) interactions are also known to regulate colonic restitution [23,24]. This led us to determine whether Sb supernatant (SbS) has an impact on cell migration via a a2b1 integrin-independent mechanism. In the present study, we have shown that SbS contained one or more heat-sensitive molecules which blocked the avb5-mediated cell-Vn interaction. This change in cell-ECM adhesion could be responsible for the observed increase in cell restitution. Methods Cell culture The human colonic adenocarcinoma cell line HCT-8/E11 (gift of Pr M. Bracke-Ghent, Belgium) was routinely cultured as previously described [25]. Cells were cultured on plastic dishes until they reached confluency. These cellular monolayers consisted of polarized cells joined by tight junctions, which exhibited well developed apical microvilli, allowing the study of processes involved in intestinal epithelial cell physiology. The human colonic adenocarcinoma cell line HT29-D4, established in our laboratory, was cultured as previously described [15]. Yeast culture supernatant Lyophilized Sb was provided by Biocodex laboratories (Gentilly, France). SbS was prepared as described previously [15,26]. In brief, Sb (100 mg/ml) was rehydrated in epithelial cell culture media RPMI 1640 without fetal calf serum and incubated overnight at 37uC in aerobiosis condition. Conditioned media were centrifuged at 20,0006 g for 15 min to separate the yeast cells from the supernatant and the supernatant collected. The supernatant was passed through 0.22 mm filters (Fisher Scientific) to remove cell debris. Serial dilutions ranging from 1/8 to 1/128 were performed in RPMI. None of the diluted supernatants affected cell viability, as verified by the trypan blue exclusion test. In some experiments, SbS was fractionated through a Pierce concentrator 9 kDa MWCO filter (Thermo Scientific). To confirm that the filtration discriminates between molecules superior and Sb Modulates avb5 Integrin Activity inferior to 9 kDa, fractions were analyzed by SDS PAGE. We never detected .9 kDa molecules in the ,9 kDa fraction (not shown). Cell adhesion assay Adhesion substrata were prepared by coating flat-bottom 96- well microtiter plates overnight at 4uC with 50 ml of vitronectin (Vn) or fibronectin (Fn) diluted in phosphate-buffered saline (PBS) at the indicated concentrations. Coated wells were blocked with 1% BSA in PBS for 30 min, and then washed twice with PBS. HCT-8/E11 single-cell suspensions (50,000 cells/0.1 ml) prepared in DMEM containing 0.2% BSA (adhesion buffer) were seeded in substratum-coated wells and allowed to adhere for 2 h at 37uC. Unattached cells were removed by 4 gentle washes with adhesion buffer and residual attached cells were fixed by 1% glutaralde- hyde. After staining with 0.1% crystal violet, cells were lyzed with 1% SDS and the optical density was measured at 600 nm by a microplate reader. When required, 10 mg/ml of function-blocking anti-integrin mAbs (rat anti- av subunit, clone 69.6.5; Beckman Coulter or mouse anti -a5 subunit, clone SAM-1; Millipore) were added during the time of cell adhesion. Cell spreading assay 24-well plates were coated at 4uC overnight with 250 ml of either Vn or Fn at 3.15 mg/ml. Coated wells were blocked with 0.5% BSA in PBS for 30 min and then washed twice with PBS. HCT-8/E11 single-cell suspensions (25,000 cells/0.5 ml) were seeded in substratum-coated wells and allowed to adhere for 2 h at 37uC. Cells were not washed in order to preserve both attached and unattached cells. The ratio of cells that have filopodia and/or lamellipodia over total cells was calculated by counting cells microscopically and was referred to the percentage of cell spreading. Cell spreading was determined by counting cells in 5 microscopic fields per well. Flow cytometry analysis HCT-8/E11 cell surface expression of integrins was determined by flow cytometry as previously described [27]. Briefly, sub- confluent cells were harvested and resuspended in DMEM containing 20% fetal calf serum and 1% BSA. The single cell suspension (106 cells/ml) was incubated for 1 h at 4uC in the presence of 10 mg/ml anti-integrin mAbs (rat anti -a6 subunit: clone GOH3, Beckman Coulter; rat anti-av subunit: clone 69-6-5, Beckman Coulter; mouse anti-b1 integrin: clone K20, Beckman Coulter; mouse anti avb5 clone P1F6, Millipore). Cells were rinsed once with ice-cold DMEM containing 0.1% BSA and then incubated for 45 min at 4uC with the appropriate Alexa Fluor 488-conjugated antibody. After washing, cells were fixed with 2% formaldehyde and cell-bound fluorescence was quantified using a Becton-Dickinson FACScan flow cytometer. Non-specific labeling was determined by incubating cells with the secondary Alexa 488- conjugated Ab alone. Focal adhesion labeling HCT-8/E11 cell suspensions (50,000 cells/0.5 ml) were either treated with or without SbS and seeded in Vn or Fn-coated wells (3.15 mg/ml). They were allowed to adhere for 2 h at 37uC. Cells were fixed for 20 min at 37uC with 3% formaldehyde in PBS, permeabilized by incubation in PBS/0.1% saponin for 30 min, then incubated for 1 h in PBS containing 4% BSA (w/v). av integrin subunit, vinculin and tyrosine phosphorylated proteins were detected with 10 mg/ml AMF-7 (Beckman-Coulter), hVIN-1 (Sigma), or PY20 (Millipore) Abs, respectively. After 4 washes in PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45047