Polyphosphate Maintains Intestinal Homeostasis Figure 3. HSP27-inducible fraction separated from culture supernatant of L. brevis 8803 was neither protein nor polysaccharide. (A) Amino acid analysis after acid degradation of HSP27-inducible fraction by HPLC. (B) Neutral sugar and uronic acid content in the fraction. (C) Peptidoglycan content in the fraction. Concentration of peptidoglycan was measured by SLP test. (D) Western blot analysis of HSP27 induction from Caco2/BBE cells by LPS derived from E. coli or Salmonella enterica, or LTA derived from B. subtilis or Staphylococcus aureus (concentration: 0.1–100 mg/mL). doi:10.1371/journal.pone.0023278.g003 between cytoskeletal components, in particular the microfila- ments inside the cell and ECM. They also play a role in cell signaling, defining cellular shape and mobility, and regulating the cell cycle. However, the role of integrins in host–microbial interactions had been unknown. While functional molecules produced by bacteria which mediate host–microbial interactions are recognized by toll-like receptors [32,33] or transporters [7,34], the present study is the first to propose that integrins are novel sensors for the probiotic-released molecules contributing to the beneficial function of probiotics. P38 MAPK is a class of mitogen-activated protein kinases responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and is involved in cell differentiation and apoptosis. It has been reported that an intraperitoneal injection of the p38 MAPK-specific inhibitor FR167653 aggravated 3% DSS-induced colitis [35]. This suggests that activation of the p38 MAPK pathway is a protective reaction against the excess inflammation. Other strains of lactobacillus including LGG also stimulated the phosphorylation of p38 MAPK [36], thus suggesting that the activation of p38 MAPK is a pivotal process in the exhibition of the beneficial functions of such bacterial strains. We also confirmed that poly P content secreted from the bacterium varied in the culture time (Figure 4D). In addition, positive relationship between secreted poly P content and HSP27 induction by the culture supernatant was observed. L. brevis began to accumulate poly P during its exponential growth phase and reached a maximum when the cells were in the early stationary phase. Similarly, it has been reported that poly P was accumulated during the growth phase in the case of Escherichia coli [37], suggesting that secretion and accumulation of poly P is dependent on intestinal condition. In unsuitable conditions due to the propagation of pathogenic bacteria or administration of antibiot- ics, the ability to produce or secrete poly P by probiotics and commensal bacteria is thought to decrease, thus leading to failed maintenance of intestinal homeostasis and the development of intestinal disorders. It is also probable that the probiotic effect on intestinal disorders is not stable when live bacteria are used. The administration of poly P rather than live bacteria is expected to exert beneficial functions regardless of the condition of the gastrointestinal tract. Moreover, unraveling the mechanism of poly P accumulation and secretion by intestinal bacteria may lead to the development of novel strategies to prevent and/or treat intestinal disorders. In a DSS-induced acute experimental colitis model, intrarectal administration of poly P significantly improved the survival rate and ameliorated intestinal injury (Figure 9A–C). Intrarectal administration of poly P also suppressed the production of inflammatory cytokines IL-1b and IL-6 in mice colons with DSS-induced colitis (Figure 9D). In our ex vivo loop assay, poly P improved the intestinal barrier function (Figure 5A). Furthermore, staurosporine-induced caspase-9 and -3 activation and TNF-a- induced NF-kB pathway activation were inhibited by pretreat- ment with the HSP27-inducible fraction (Figures 2 and 8). Accordingly, both the cytoprotective and anti-inflammatory properties of poly P probably contribute to improve the intestinal injury caused by DSS-induced colitis. It has been reported that poly P is essential for the survival of pathogenic bacteria such as Salmonella, Shigella, Pseudomona aeruginosaand, and Neisseria meningitidis, which produce virulent factors [38–41]. However, it is unclear whether poly P itself is a virulent factor. In our ex vivo loop study and DSS-induced mouse colitis model, poly P showed beneficial functions without any adverse effects. However, it remains unclear whether pathogenic bacteria-derived poly P is secreted into its living environment, while some probiotic bacteria are thought to secrete poly P in the culture supernatant as shown in this study. Therefore, poly P produced by pathogens appears to be used as an intracellular energy source, whereas probiotic-derived poly P can be released and can exert multiple functions including a cytoprotective effect. Further analysis of the mechanism by which probiotics secrete poly P from the cytoplasm to the extracellular environment is needed to discriminate the functions of probiotic-derived poly P from those of pathogen-derived poly P. In summary, the present study showed that poly P produced by probiotics was a bioactive molecule that induced cytoprotective HSP through activation of the integrin–p38 MAPK pathway, and prevented oxidant-induced intestinal barrier weakening. The integrin–p38 MAPK pathway is a novel system for sensing the bioactive components secreted by probiotics and commensal bacteria. Furthermore, poly P ameliorated epithelial injury in the colon of mice with DSS-induced colitis, illustrating that poly P is a promising agent for preventing and alleviating intestinal inflam- mation. Determining the secretory mechanism of poly P by probiotic and/or commensal bacteria essential for maintaining intestinal homeostasis will lead to a better understanding of the pathogenesis of inflammatory disorders and aid future therapeutic development. Materials and Methods Microorganisms L. brevis SBC8803 and SBC8013 used in this study was supplied by Sapporo Breweries Ltd. LGG was purchased from the American Tissue Culture Collection (ATCC). These lactobacilli were cultured in de Man–Rogosa–Sharpe (MRS) broth (Difco Laboratories, Detroit, MN) for a day at 37uC. Cell culture Human colonic epithelial Caco2/BBE cells were purchased from ATCC. They were grown in high-glucose Dulbecco’s Modified Eagle’s medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin and 10 mg/mL transferrin (all from Invitrogen/GIBCO, Grand Island, NY) in a humidified atmo- sphere of 5% CO2. The cells were plated on 6- or 12-well plates at a density of 105 cells/cm2 and then allowed to differentiate for 10– 14 days before experiments. Mice The studies were approved by the Institutional Animal Care and Use Committee of the Asahikawa Medical University (the permit number 10080). C57Bl/6 mice were purchased from Sankyo Labo Service (Tokyo,Japan). PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23278