S. boulardii Enhances Cell Restitution Figure 4. Sb supernatant but not Sc supernatant improves mouse enterocyte migration along the crypt-villus axis. Mice were daily force-fed for one week with unused culture medium for Sb (control), Sb supernatant (Sb sn) or Sc supernatant (Sc sn) then 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, B and C) and quantitatively assessed (D) as described in Methods. (n = 5). ** P,.01. Arrow: basal site of the enterocytes. Scale bar: 150 mm. doi:10.1371/journal.pone.0018427.g004 Sb supernatant increases a2b1 integrin activation state We postulated that Sb supernatant exerts its effects by activating the a2b1 integrin. To test such a possibility, we activated the a2b1 integrin using TS2/16, a b1 integrin activating mAb [28]. As expected, a2b1 activation increased cell Figure 5. a2b1 integrin is relocalized in the intestine of Sb force-fed mice. Intestinal tissues from control (A and C) or Sb force- fed mice (B and D) were immunostained with Abs against a6 (A and B) or a2 integrin (C and D). Each image is a representative image taken from tissue sections of five mice. Scale bar: 130 mm. doi:10.1371/journal.pone.0018427.g005 adhesion onto type I collagen (Figure 8A). This activation also led to an increase in cell migration such as that observed with Sb supernatant (Figure 8B). Pretreatment of cells with integrin a2b1 activating factor improved neither Sb-stimulated cell adhesion nor cell restitution (Figure 8A and 8B), strongly suggesting that Sb secretes factors that activate a2b1 integrin, either directly or indirectly. Sb regulates the a2b1 integrin signaling pathway After activation, integrins quickly associate within the actin cytoskeleton and cluster together to form focal adhesions, multimolecular complexes that contain signaling molecules including focal adhesion kinase (FAK), paxillin and Erk1/2. Activation of these signaling molecules leads to the modulation of cell migration. We therefore checked the phosphorylation status of these different key signaling molecules. As observed in Figure 9A, cell adhesion to type I collagen promoted phosphorylation of both FAK and paxillin on the Y397 and Y118 tyrosine residues respectively. Interestingly, Sb supernatant increased the tyrosine phosphorylation level of both proteins. Sb also upregulated the tyrosine phosphorylation status of Erk1/2, another protein associated with integrin signaling pathways (Figure 9A). We next tested the effect on cell migration of PD 98059 and PF 573228, inhibitors of MAPK and FAK respectively. As observed on Figures 9B and 9C, these inhibitors fully blocked the Sb-induced cell migration. These results confirm that Sb stimulates cell migration through the activation of multiple signaling pathways including activation of FAK and Erk1/2. PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e18427