S. boulardii Enhances Cell Restitution Figure 3. Sb improves mouse enterocyte migration along the crypt-villus axis without affecting crypt cell proliferation. Control and force-fed Sb mice were injected with BrdU 24 hours before sacrifice. Small intestinal tissues were processed for immunostaining. The BrdU-labeled enterocyte migration along the crypt-villus axis was traced on tissue sections by immunostaining with an anti-BrdU Ab (A and B) and quantitatively assessed (C) as described in Methods. (n = 5). ** P,.01. The proliferative marker ki-67 was detected in small intestine of control (D) or Sb force-fed mice (E) and the number of ki-67 positive cells per crypt were evaluated (F) (n = 5). Scale bar: 150 mm. doi:10.1371/journal.pone.0018427.g003 were detected mainly within the crypts in both control and Sc supernatant force-fed mice. However, in Sb supernatant force-fed mice, BrdU positive cells were distributed from the crypt to the tips of the villi (Figures 4B and 4D). These data suggest that Sb supernatant, but not Sc supernatant, contains molecule(s) that induce migration of enterocytes in vivo. a2b1 integrin collagen receptor is involved in Sb-dependent cell restitution Enterocyte migration requires dynamic interactions between ECM molecules, such as laminin isoforms and collagens, and their cell surface receptors [27]. Therefore we determined whether Sb affected the distribution of a2 and a6 integrin subunits, receptors of collagens and laminin, respectively. In both control and Sb force-fed mice, a6 was detected at the basolateral surfaces of enterocytes on the villus and in the crypt (Figure 5A and 5B). In control mice, a2 integrin was found mainly at the lateral sites of the enterocytes whereas it was significantly redistributed to the basal sites in Sb force-fed mouse cells (Figure 5C and 5D). These results suggest that a2 integrin participates in Sb-enhanced enterocyte migration. To confirm the role of a2b1integrin in Sb-enhanced enterocyte migration, HCT-8/E11 cells, which express the integrins a2b1, a3b1, a5b1, avb5, and a6b4 (unpublished data), were incubated with function blocking anti-integrin mAbs during cell migration. As observed in Figure 6A, in control cells only the anti-av integrin mAb inhibited the capacity of untreated HCT-8/E11 cells to migrate by approximately 50%. Interestingly, Sb-dependent cell migration was also inhibited by the anti-av mAb (70%), and additionally with the anti-a2 (40%) mAb. Altogether these data indicate that the Sb-induced enterocyte migration depends on a2b1 integrin. It should be noted that incubation with a mixture containing anti-av, -a6 and -b1 integrin mAbs did not totally abolish cell migration in either condition, suggesting the participation of integrin-independent cell adhesion molecules in this process. Sb modulates a2b1 integrin-mediated adhesive properties As adhesion is essential for cell migration, we next examined whether treatment with Sb supernatant could alter HCT-8/E11 cell attachment to ECM proteins. Cells incubated with or without Sb supernatant were allowed to attach to increasing concentrations of purified laminin-111, or type I collagen. HCT-8/E11 cells attached to both matrices (Figure 6B). Sb supernatant did not alter cell adhesion to laminin-111. However, it dramatically increased the percentage of adherent cells on the type I collagen matrix. Adhesion assays performed in the presence of anti-integrin function blocking mAbs revealed that a2b1 integrin is the sole receptor for type I collagen in these cells (Figure 7). Thus, the increase in cell adhesion to type I collagen upon Sb treatment most likely occurs through the modulation of the a2b1 integrin functional state. PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e18427