tissues from oxidant stress and improve the intestinal injury in a mouse colitis model [13]. We tried to isolate the cytoprotective compound from the culture supernatant of L. brevis SBC8803. In this study, we proposed for the first time that polyphosphate (poly P), a common product of lactobacilli and other bacteria associated with intestinal microflora, is the molecule responsible for probiotic actions. In addition, we revealed that intestinal integrin b1medi- ated the bacteria-derived poly P functions including enhancement of intestinal barrier function and improvement of intestinal injury in mouse colitis model. Results Induction of cytoprotective HSP27 by the culture supernatant of L. brevis SB8803 in Caco2/BBE cells Human intestinal epithelial Caco2/BBE cells were stimulated with the culture supernatant of L. brevis SBC8803 in vitro to elucidate HSP27 induction. The culture supernatant was eluted by filtration with a 0.2 mm filter to completely remove the bacterial body from culture medium. HSP27 was induced by the culture supernatant while constitutive HSC70 expression remained unchanged. HSP27 induction was affected by the bacterial culture time (Figure 1A). The supernatant obtained at 12 h from bacterial culture (0.116109 cfu/mL) strongly induced HSP27 compared with that obtained at 36 h (1.076109 cfu/mL) and 60 h (0.956109 cfu/mL). Similar results were obtained in the case of LGG and L. brevis SBC8013 (Figure 1B). Identification of the molecule responsible for the induction of HSP27 To help identify the molecule in the culture supernatant of L. brevis SBC8803 responsible for HSP27 induction from Caco2/ BBE cells, we first investigated the molecular weight of the HSP27- inducible molecule. The culture supernatant was separated by spin columns equipped with 5-, 10-, 30- or 50-kDa molecular weight cutoff (MWCO) membranes. HSP27 induction by filtrates passed through the 5- and 10-kDa MWCO membranes was diminished, but remained unchanged in filtrate passed through 30- and 50- kDa membranes (Figure 1C). These results indicated that the HSP27-inducible molecule had a relatively high molecular weight. In addition, the culture supernatant was precipitated by ammonium sulfate [(NH4)2SO4]. At 65% (NH4)2SO4 saturation, the precipitate strongly induced HSP27 (Figure 1D). This precipitate was further separated by diethylaminoethyl (DEAE) anion-exchange chromatography. The fraction eluted with 1.0 M sodium chloride strongly induced HSP27 (Figure 1E). This fraction was further separated by size-exclusion chromatography (Figure 1F). The HSP27-inducible fractions were collected and separated by high-pressure liquid chromatography (HPLC) and finally a single peak fraction was obtained (Figure 1G).This fraction significantly induced HSP27 (Figure 1H). Because HSP27 has been shown to inhibit cytochrome c-mediated activation of caspases [14], the inhibitory effect of this fraction was investigated using staurosporine-induced apoptosis in caco2/BBE cells. Cas- pase-9 and -3 activation were inhibited by this fraction (Figure 2). The molecular weight of this fraction was relatively high and had strong adsorption on the DEAE anion-exchange resin. Thus, we assumed that this fraction was protein and investigated the amino acid composition of this fraction by acid hydrolysis; however, no amino acids were detectable (Figure 3A). Further- more, the total content of neutral sugar and uronic acid measured in the acid hydrolysate was ,1% (Figure 3B) and the peptideglycan content determined by a silkworm larvae plasma test was extremely low (Figure 3C). Collectively, these results Polyphosphate Maintains Intestinal Homeostasis indicated that the HSP27-inducible fraction was neither a protein nor a polysaccharide. Bacterial cell wall lipopolysaccharide or lipoteichoic acid did not induce HSP27 from Caco2/BBE cells (Figure 3D). Unexpectedly, the purified material contained a large amount of phosphorus and oxygen; therefore, we measured the poly P contents by toluidine blue O (TBO) and molybdenum blue methods. The phosphoric acid content after acid hydrolysis was 0.92 mg/mg, (.90% w/w). The decrease in 620 nm absorbance of a 1 mg/mL solution caused by the metachromatic reaction with TBO was 0.097. Based upon these results, we assumed that the HSP27-inducible fraction was poly P. To confirm this supposition, poly P was synthesized by poly P- synthesizing enzyme polyphosphate kinase (PPK) and purified by HPLC (Figure 4A). The synthesized poly P significantly induced HSP27 from Caco2/BBE cells in a dose-dependent manner (Figure 4B). Furthermore, to confirm that poly P in the culture supernatant of L. brevis was responsible for HSP27 induction from Caco2/BBE cells, the ability to induce HSP27 by poly P was investigated when poly P in the HSP27-inducible fraction was degraded. Enzymatic reaction by PPK is reversible and usually favors poly P synthesis. If the reaction solution contains a large amount of adenosine diphosphate (ADP) in comparison to adenosine-59-triphosphate (ATP), poly P is degraded to equilibrate the molecular ratio of ADP/ATP. After the treatment of PPK with a large amount of ADP (10 mmol/l), the HSP27-inducible fraction was unable to induce HSP27 production from Caco2/BBE cells (Figure 4C). This result suggests that poly P is the molecule responsible for induction of HSP27. Moreover, we confirmed that poly P content in the culture supernatant of lactobacilli varied in the culture time (Figure 4D). Improvement of the epithelial barrier function by Poly P through mediation of integrin–p38 MAPK pathway To determine the physiologic consequences of poly P effects, transmural [3H]-mannitol fluxes were measured in intact small bowel loops to assess the intestinal barrier function. Increased mucosal permeability in small intestinal loops caused by exposure to an oxidant (NH2Cl, 0.3 mM) was significantly inhibited by 10 mg/mL of luminal poly P (Figure 5A). Phosphorylated HSP27 protects the actin cytoskeleton from depolymerization by inter- acting with actin [15,16]. We confirmed that cytochalasin D, which severs actin filaments [17], inhibited the protective action by poly P (Figure 5F). Furthermore, poly P treatment of Caco2/ BBE cells enhanced F-actin stability and attenuated its degrada- tion induced by treatment with 30 mM hydrogen peroxide (Figures 6A, B), 0.3 mM of monochloramine (NH2Cl) (Figures 6G, H). To determine the signal transduction pathway for inducing HSP 27, the activation of the representative pathways including p38 MAPK, extracellular signal-regulated kinase (ERK), c-Jun N- terminal kinase (JNK) and Akt was examined. P38 MAPK was phosphorylated by poly P, while phosphorylation of ERK, JNK and Akt were not detected (Figure 7A). Then, the mediation of p38 MAPK to protect the intestinal epithelia against oxidant stress was investigated. The protective action against oxidant stress by poly P was inhibited by addition of the p38 MAPK inhibitor SB203580 (Figure 5B), indicating that the protective effect of poly P was mediated by activation of p38 MAPK. Poly P appeared not to pass the tight junction of epithelial cells due to its high molecular weight. It was reported that integrins, which are a family of receptors that mediate cell adhesion to extracellular molecules, were associated with p38 MAPK [18]. We confirmed by immunohistochemical staining that integrin b1 and b3 molecules were apically expressed (Figure 7B). Peptide PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23278