ylation may influence mucosal wound healing [44]. Therefore, we suggest that SbS regulates the strength of the avb5 integrin/Vn interaction and consequently modulates FAK tyrosine phosphor- ylation, leading to a change in avb5 integrin-dependent migration. Changes in cell-ECM interactions occur spatially and tempo- rally during intestinal wound healing. Several in vivo and in vitro studies in the gut indicate the requirement of the binding and interaction of ECM-specific integrins for this restitution, such as a3b1, a6b1, a6b4 laminin-binding integrins, a2b1 collagen- binding integrin or av-subunit Fn-binding integrin [7,45]. We recently provided data indicating that Sb exerts at least some of its motogenic effect through the activation of the collagen receptor a2b1 integrin [15]. Here we have reported that SbS also exerts some of its motogenic effect via down-modulation of the interaction between avb5 integrin and Vn. Indeed, (1) inhibition assays using anti-integrin mAbs demonstrated that avb5 integrins participate in the SbS-induced cell migration. Given the ligand- binding properties of these integrins, it appears likely that Vn supports this process. (2) Both cell adhesion and cell spreading to Vn, two processes required for cell migration, were altered by SbS. (3) Sb promoted the reorganization of avb5 integrins into adhesive structures localized at the leading edge of the cells. These are crucial to migration and are themselves regulated by SbS. Moreover, SbS was shown to promote the redistribution of the Vn receptor in the mouse intestine. This redistribution was observed for both epithelial and non-epithelial cells. In line with these data, some studies have indicated a crucial role of Vn in regulating cell migration during wound healing [46,47]. Cell migration requires the dynamic interaction between cells and the substratum on which they are attached and over which they migrate [48]. Changes in the density of ligand, integrin repertoire, ligand-binding affinity and/or cytoskeletal associations are all key determinants of cell migration speed [48,49]. In the present study, we showed, by flow cytometry analysis, that SbS did not alter the concentration of avb5integrin or, in previous work, a2b1integrin [15]. However, our data strongly suggest that SbS dynamically regulates the assembly and disassembly of adhesions that are essential for optimum cell migration. Indeed, to promote cell restitution, SbS activates the a2b1 integrin collagen receptor [15] whereas it down-regulates the av integrin interactions with Vn (this work). Sb acts as a shuttle that could liberate, during the intestinal transit, at least 1500 molecules that have not been totally characterized [50]. This large number of secreted peptidic and non-peptidic factors, including proteases, phosphatases and polyamines, may at least partially explain why Sb has pleiotropic effects on intestinal mucosa and also has therapeutic effects on such a wide variety of gastrointestinal disorders [14,31,51]. Some of the molecules, as yet unidentified, can interfere with host cell signaling pathways and are therefore able to modulate host cell References 1. Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J (2007) Restoration of barrier function in injured intestinal mucosa. Physiol Rev 87: 545–564. 2. Mumy KL, Chen X, Kelly CP, McCormick BA (2008) Saccharomyces boulardii interferes with Shigella pathogenesis by postinvasion signaling events. Am J Physiol Gastrointest Liver Physiol 294: G599–609. 3. Martins FS, Dalmasso G, Arantes RM, Doye A, Lemichez E, et al. (2010) Interaction of Saccharomyces boulardii with Salmonella enterica serovar Typhimurium protects mice and modifies T84 cell response to the infection. PLoS ONE 5: e8925. 4. Sturm A, Dignass AU (2008) Epithelial restitution and wound healing in inflammatory bowel disease. World J Gastroenterol 14: 348–353. 5. Swanson PA 2nd, Kumar A, Samarin S, Vijay-Kumar M, Kundu K, et al. (2011) Enteric commensal bacteria potentiate epithelial restitution via reactive oxygen species-mediated inactivation of focal adhesion kinase phosphatases. Proc Natl Acad Sci U S A 108: 8803–8808. Sb Modulates avb5 Integrin Activity behavior including intestinal mucosal inflammatory, secretory, and barrier functions [50]. Moreover, Sb produces other factors that reduce inflammation by blocking NF-kB and MAPK activation [26,52] and enhancing PPAR-c expression [53]. On the other hand, conditioned medium of Sb was shown both in vitro and in vivo to modulate host signaling pathways involved in the regulation of cell motility including the MAPK and FAK pathways [15,26]. Although the motogenic molecule(s) secreted by Sb remain to be elucidated we have reported here that SbS contains two groups of heat-sensitive compounds capable to increase intestinal restitution: one group with a molecular weight lower than 9 kDa and another with a molecular weight higher than 9 kDa. We can postulate that this latest class of molecules corresponds to the same as those involved in the down-modulation of cell adhesion to Vn. In conclusion, this report demonstrates that SbS contains various heat-sensitive motogenic factors that can improve intesti- nal restitution. These factors exerted their effect through multiple pathways, including the dynamic fine regulation of integrin- mediated adhesion to the ECM. This could be of major importance in diseases characterized by severe mucosal injury, as seen in IBD or infectious gastroenteritis. Supporting Information Video S1 HCT-8/E11 cell monolayers were wounded as described in Methods, incubated with Sb supernatant (dilution 1/8), then 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 h, using a Cool SnapHQ camera (Princeton Instrument) through a 106 objective lens. (MOV) Video S2 HCT-8/E11 cell monolayers were wounded as described in Methods and incubated with SbS. Cell monolayers were further incubated with or without function- blocking anti- av integrin mAb 1 h before wounding and during cell migration. 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 time of 5 h using a Cool SnapHQ camera (Princeton Instrument) through a 106 objective lens. (MOV) Author Contributions Conceived and designed the experiments: FA VR DL. Performed the experiments: AC EP CS DC CT. Analyzed the data: FA VR SB EP AC DL JM. Contributed reagents/materials/analysis tools: DC. Wrote the paper: FA. 6. Goke M, Kanai M, Lynch-Devaney K, Podolsky DK (1998) Rapid mitogen- activated protein kinase activation by transforming growth factor alpha in wounded rat intestinal epithelial cells. Gastroenterology 114: 697–705. 7. Basson MD (2001) In vitro evidence for matrix regulation of intestinal epithelial biology during mucosal healing. Life Sci 69: 3005–3018. 8. Zhang J, Owen CR, Sanders MA, Turner JR, Basson MD (2006) The motogenic effects of cyclic mechanical strain on intestinal epithelial monolayer wound closure are matrix dependent. Gastroenterology 131: 1179–1189. 9. Basson MD, Modlin IM, Madri JA (1992) Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J Clin Invest 90: 15–23. 10. Basson MD, Rashid Z, Turowski GA, West AB, Emenaker NJ, et al. (1996) Restitution at the cellular level: regulation of the migrating phenotype. Yale J Biol Med 69: 119–129. 11. Gilcrease MZ (2007) Integrin signaling in epithelial cells. Cancer Lett 247: 1–25. PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e45047