S. boulardii Enhances Cell Restitution Figure 6. Sb-stimulated cell restitution requires modulation of the interaction between a2b1 integrin and collagen. (A) HCT-8/E11 cell monolayers were wounded and incubated with or without Sb supernatant. The monolayers were further incubated with or without (2) function- blocking anti-integrin mAbs (10 mg/mL) 1 hour before wounding and during cell migration. Wound closure was determined as described in Methods. Results are expressed as the percentage of cell migration compared to control. Data represent the mean 6 SD of 5 separate experiments. ** P,.01; * P,.05. A mouse anti-DPP IV (DPPIV) mAb was used as a control for anti-integrin mAb specificity. (B) Isolated HCT-8/E11 cells were treated with or without Sb supernatant and plated on either laminin-111 or type I collagen at the indicated concentrations. Cell-ECM adhesion was evaluated as described in Methods. Results are expressed as the percentage of cell adhesion. Data represent the mean 6 SD of 3 separate experiments. doi:10.1371/journal.pone.0018427.g006 We analyzed the impact of Sb supernatant on the organization of adherence structures (Figure 10). Paxillin staining revealed that control cells, plated on type 1 collagen, exhibited few focal adhesion structures. However paxillin staining in Sb treated cells displayed more focal adhesion structures that appeared to be thicker than those observed in the control condition (Figure 10A and 10B). Staining of focal adhesion structures on migrating cells yielded a similar pattern (Figure 10C and 10D). This indicates that treatment with Sb supernatant is associated with functional and structural modifications of focal adhesion structures. To explore whether Sb also regulates focal adhesion structures in vivo, we analyzed the paxillin tyrosine phosphorylation status on intestine from mice force-fed with PBS alone, or PBS containing 1 mg/ml lyophilized Sb. As depicted in Figure 10E, an anti-Y118 paxillin Ab failed to stain any structure in control C57BL6J mice. However, in Sb force-fed mice, staining was detected, confined to the basal surface of the enterocytes (Figure 10F). This strongly suggests that Sb promotes the activation of adhesion structures found in enterocytes in vivo. Taken together these data indicate that Sb secretes factors that strengthen a2b1/collagen interactions leading to enhanced outside-in signaling and focal adhesion structure reorganization. This was associated to an increase in cell migration. Discussion Besides a marked infiltration of inflammatory cells, epithelial cell damage is also frequently observed in the mucosal lesions of IBD such as ulcerative colitis, Crohn’s disease or in infectious gastroenteritis [2]. Therefore, complete remission of such diseases requires both the cessation of inflammation and the repair of damaged epithelium. The development of novel therapies that accelerate the repair of intestinal epithelium has recently begun and various molecules are now being considered for clinical use including epidermal growth factor in combination with mesala- mine [29], keratinocyte growth factor [30], and hepatocyte growth factor [31]. However, a better understanding of the biological effects of these molecules must be ascertained, not least to identify any undesired secondary effects, such as tumorigenesis. In the present study, we analyzed the effect of Sb on the capacity of intestinal epithelial cells to reseal a wound. According to our findings, the impact of Sb on epithelial cells can be summarized as PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e18427