Polyphosphate Maintains Intestinal Homeostasis Figure 8. Anti-inflammatory property of HSP27-inducible fraction. Time course of NF-kB pathway activation in Caco2/BBE cells induced by 10 ng/mL TNF-a treatment in the presence or absence of 10 mg/mL of poly P. doi:10.1371/journal.pone.0023278.g008 ized to GAPDH expression (sense, 59-tgtgtccgtcgtggatctga-39, anti- sense, 59-ttgctgttgaagtcgcaggag -39 for the analysis of mouse GAPDH,). Serial dilutions of a standard solution were included for each gene to generate a standard curve and allow calculation of the input amount of cDNA for each gene. A LightCycler 480 optical capillary tube was incubated at 95uC for 10 min to activate the FastStart Taq DNA polymerase. The run condition were 45 cycles at 95uC for 10 s, 59uC for 20 s, and 72uC for 50 s. Melting curves of each amplified gene were created to obtain PCR efficiency. Ex vivo intestinal loop studies C57Bl/6 mice were sacrificed and the small intestine was removed beginning at the ligament of Treitz. The small intestine was divided into 3–6 pieces, each end ligated with silk sutures and the loops filled with RPMI 1640 medium containing sample. Loops were incubated for 2 h at 37uC in a 5% CO2 incubator. To measure permeability effects, the loops were filled with RPMI 1640 medium containing 1 mCi/mL [3H]-mannitol with or without 0.3 mM freshly prepared monochloramine (NH2Cl). Loops were placed into the middle section of the organ culture dish in 4 mL of RPMI 1640 without NH2Cl. RPMI 1640 mediums in the culture dish were taken at 5, 20 and 35 min to determine the flux of mannitol from the lumen to the medium outside bathing loops that were being bathed. Induction and assessment of colitis C57Bl/6 mice were administered 3% (wt/vol) DSS (MW, 2500 Da) in the drinking water. In the test group (n = 5), 10 mg of L. brevis-derived poly P dissolved in 100 mL of PBS was intrarectally administered once a day throughout the experimental period. In the control group (n = 5), 100 mL of PBS was intrarectally administered once a day throughout the experimental period. The mice were sacrificed on the 7th day following DSS treatment and the entire colon was removed from the cecum to the anus, and the colon length was measured as a marker of inflammation. Gene expression in the colon was investigated using real-time RT-PCR. In another experiment, the survival rate of 4% DSS-administered mice was investigated. Poly P-integrin interaction assay To synthesize 32P-labeled poly P, 1 mL of reaction mixture containing 50 mM Tris-HCl (pH 7.4), 40 mM ammonium sulfate, 4 mM MgCl2, 40 mM creatine phosphate, 20 ng/mL creatine kinase, 1 mM ATP (pH 7.2), 100 mCi of [c-32P]ATP and 1 U of polyphosphate kinase (PPK) from Propionibacterium shermanii was incubated for 3 h at 37uC, and the reaction was stopped by adding 100 mL of 50 mM CaCl2 and then centrifuged at 50006g for 10 min. The precipitates were dissolved by adding 50 mM ethylenediaminetetraacetic acid (EDTA) solution, and then low- molecular weight components such as Ca2+ ions and EDTA were removed from the solution by dialysis using a tube equipped with 3-kDa MWCO membrane. Twenty ml of 100 mg/ml human integrin a3b1 or aVb3 was added into 50 ml of 32P-labeled poly P solution and reacted for 1 h at room temperature. Reaction mixtures were immunoprecipitated with 1 mg of anti-human integrin b1 antibody, mouse IgG1 isotype control antibody, anti- human integrin b3 antibody, or goat IgG isotype control antibody (R&D systems) and 20 ml of Bio-Adembeads Protein G beads (Ademtech, Pessac France). After 1 h incubation at room temperature, immunoprecipitated 32P-labeled poly P was deter- mined by liquid scintillation counting. Histological evaluation of colitis The colons were fixed in 10%-buffered formalin solution, embedded in paraffin using standard methods, cut into 5-mm sections, stained with H&E, and then assessed under light microscopy. The histological activity was assessed according to Berg’s score described below [42]. The grades of the intestinal inflammation were assessed in three representative parts of the colon in each mouse because of multi focal and variable severities of the intestinal lesions. A score from 0 to 4 was based on the following criteria: (grade 0) no change from normal tissue; (grade 1) one or a few multi focal mononuclear cell infiltrates in the lamina propria accompanied by minimal epithelial hyperplasia and slight to no depletion of mucus from goblet cells; (grade 2) the lesions tended to involve more of the intestine than grade 1 lesions, or were more frequent. Typical changes included mild inflamma- tory cell infiltrates in the lamina propria composed primarily of mononuclear cells with a few neutrophils. Small epithelial erosions were occasionally present and inflammation rarely involved the submucosa; (grade 3) lesions involved a large area of mucosa or were more frequent than grade 2 lesions. Inflammation was moderate and often involved the submucosa but was rarely transmural. Inflammatory cells were a mixture of mononuclear cells as well as neutrophils, and crypt abscesses were sometimes observed. Ulcers were occasionally observed; (grade 4) such lesions usually involved most of the intestinal section and were more severe than grade 3 lesions. Inflammation was severe, including mononuclear cells and neutrophils, and it was sometimes transmural. Crypt abscesses and ulcers were present. Immunohistochemistry Immunohistochemistry for integrin b1 and b3 were performed by using anti-integrin b1, rabbit-poly (I782, Bioworld Technology, PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e23278