Figure 7. a2b1 integrin is the sole receptor for type I collagen in HCT8/E11 cells. Isolated HCT-8/E11 cells were incubated with or without Sb supernatant in the absence (2) or presence of either anti-a2 or -a6 integrin mAbs. Cells were then seeded on type I collagen (3 mg/ ml). Cell adhesion was evaluated as described in Methods. Data represent the mean 6 SD of 3 separate experiments. ** P,.01. doi:10.1371/journal.pone.0018427.g007 follows: (1) Sb supernatant enhances the restitution of various differentiated human intestinal epithelial cell lines in vitro; (2) in the mouse intestine, Sb improves migration of enterocytes along the crypt-villus axis; (3) Sb does not influence enterocyte proliferation either in vitro or in vivo; (4) Sb stimulates a2b1 integrin, thus activating molecules such as FAK, paxillin and ERK1/2, which are associated with integrin signaling pathways and which modulate cell-type I collagen I interaction; (5) concomitant with this activation, Sb modulates both the structure and the activity of focal adhesions. Taken together, these effects could contribute to the enhancement of intestinal cell restitution. Intestinal wound healing is initiated by the migration of epithelial cells adjacent to the injured surface into the wound to cover the denuded area. This process, termed epithelial restitution, occurs within a period of minutes to hours and does not require cell proliferation [2]. Through the use of a relevant in vitro model [32], we have shown that Sb supernatant increased the capacity of differentiated enterocytes to recolonize a wound. The effect of Sb on wound healing was associated with an increase in cell motility rather than a stimulation of cell proliferation. Indeed, wound repair was unaffected by treatment with DNA synthesis inhibitors such as mitomycin C and BrdU incorporation experiments indicated that only a few proliferating cells were found in wounded areas. Also Sb did not alter cell growth (not shown). Furthermore, videomicroscopy experiments showed that Sb supernatant doubled the migration rate of HCT-8/E11 cells and Sb increased the rate of migration of enterocytes along the crypt- villus axis in mice without affecting the proliferative compartment. According to these data, we postulated that Sb supernatant contains motogenic factor(s) that improve intestinal cell restitution. Although the physiological pathways involved in intestinal restitution remain partially unexplained, it has been proposed that changes in cell-ECM interactions occur spatially and temporally during this process. Studies carried out on gut tissue revealed that ECM proteins including fibronectin, type I collagen and laminins play a crucial role in regulating cell restitution [6,7,33]. They have also demonstrated the requirement for the binding and interaction of ECM-integrins such as a3b1, a6b1, a6b4 (laminins receptors), S. boulardii Enhances Cell Restitution Figure 8. Sb modulates integrin activation state. HCT-8/E11 cells were incubated with or without Sb supernatant in the absence (2) or the presence of a b1 integrin-activating mAb (TS2/16) (A). Cells were allowed to adhere to 3 mg/ml type I collagen for 30 minutes. Cell adhesion was evaluated as described in Methods. ** P,.01. (B) Cells were allowed to migrate as described in Methods. Results are expressed as the percentage of cell migration compared to control. Data represent the mean 6 SD of 3 separate experiments. ** P,.01. doi:10.1371/journal.pone.0018427.g008 a2b1 (collagens receptor), or avb3 or avb6 (fibronectin receptors) for intestinal restitution [6,34]. In line with these results, we have provided evidence for the requirement of a2b1 integrin facilitated cell-ECM interaction for Sb-induced restitution. Sb was shown to promote the redistribution of the collagen receptor from the lateral to the basal membrane domain of enterocytes. Inhibition assays using anti-integrin mAbs demonstrated that a2b1 integrins participate in the Sb-induced cell migration, suggesting that collagens support this process. Also, adhesion assays suggested that Sb specifically modulates the a2b1-dependent cell interaction with type I collagen. The mechanisms by which Sb modulates integrin-ECM interactions and subsequent intestinal restitution need to be explored in more depth. However, at least two possibilities exist. Firstly, cell restitution could be modulated through the regulation of the integrin functional state. In accordance with this, some data have highlighted dynamic changes in integrin affinities for ligands. These changes are thought to occur in response to extracellular cues, and can modify cell migration [35]. In line with these data, we demonstrated that factors secreted by Sb can activate a2b1integrin. Indeed, cell treatment with the b1 integrin activating factor TS2/16 mAb [28,36] promoted the same increase in cell adhesion as Sb. Moreover, cell pretreatment with TS2/16 mAb did not enhance the impact of Sb on cell adhesion or cell restitution. In addition, Sb did increase a2b1 integrin signaling pathways. PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e18427