were used for performing the experiments. Cell viability was assessed using trypan blue stain prior to and following co-culture with bacteria. Cell Isolation CD4+CD25+ cells were isolated from mesenteric lymph nodes of bacteria or vehicle fed BALB/c mice to 89% purity using MACS bead mouse Treg isolation kit (Miltenyi Biotec,Au- burn,CA). CD4+CD25– T cells were isolated from DO.11–10 transgenic mice to a purity of 94%. DC were isolated from mesenteric lymph nodes of na¨ıve wild type mice using Miltenyi Biotec CD11c Microbeads. Adoptive Transfer of Dendritic Cells Bacteria were co-cultured with BMDC at a ratio of 10:1/ bacteria:DC, for 24 hr at 37uC. DC were washed three times with PBS and viability assessed with trypan blue stain prior to injection into the footpad (16106 cells). This dose was chosen on the basis of previous studies [59]. Unloaded DC and bacteria alone served as controls. T-regulatory Cell Suppressive Function To examine the suppressive capacity of JB-1 induced T- regulatory cells, we used the in vitro assay of T-regulatory cell activity as described previously [12]. Briefly, T regulatory cells (Miltenyi Biotec) were isolated from mesenteric lymph nodes of JB- 1- or vehicle-MRS broth fed mice. This cell subset (6.256103 ) were cultured with CD4+CD25– responder T cells isolated from DO11.10 transgenic mice (5 6 104 ) for 4 days in U-bottomed 96- well plates at a ratio of 1 Treg to 8 CD4+CD25– responder T cells in the presence of soluble anti-CD3 (1 mg/ml) and BM-DC (5 6 103 ). Prior to the co-culture assay, DC were pulsed with 1 mg/ml OVA protein for 16 h followed by a 20 min incubation with 50 mg/ml mitomycin C in a 37uC, 5% CO2 humidified incubator. Responder T cells were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE). Proliferation of CD4+ T cells from DO11.10 mice, detected by KJ1–26 mAb specific for the trans- genic TCR expressed by DO11.10 T cells, was assayed by CFSE dilution using FACS analysis and proliferation calculated using FloJo version 9.5.2 software (TreeStar, Ashland, OR). T cell Proliferation and Stimulation Isolated mesenteric CD11c+ DC (Miltenyi Biotec ) were co- cultured with bacteria at a ratio of 10:1/bacteria:DC, for 2 h at 37uC and after extensive washing were incubated with purified CD4+CD25– responder T cells from DO11.10 transgenic mice for 5 days in U-bottomed 96-well plates in the presence of soluble anti-CD3 (1 mg/ml) and 1mg/ml OVA protein. Where indicated, anti-IL-10 (1 mg/ml, BD Pharmingen), anti-TGFb (50 mg/ml, R&D Systems) or CrMP 5 mM was added to the culture. On day 5 References 1. Sudo N, Chida Y, Aiba Y, Sonoda J, Oyama N, et al. (2004) Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J Physiol; 558(Pt 1): 263–275. 2. Kalliomaki M, Isolauri E (2003) Role of intestinal flora in the development of allergy. Curr Opin Allergy Clin Immunol; 3(1): 15–20. 3. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, et al (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 331(6015): 337–41. 4. Geuking MB, Cahenzli J, Lawson MA, Ng DC, Slack E, et al. (2011) Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity. 34(5):794–806. 5. Round JL, Mazmanian SK (2010) Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A. 107(27): 12204–9. Lactobacillus Induced Heme Oxygenase 1 of culture T cells were harvested and replated into anti-CD3 (10 mg/ml) pre-coated 96-well cell culture plates and at a density of 16106 cells/ml with soluble anti-CD28 (2 mg/ml) for 48 hours. Supernatants were collected and stored at 280uC before cytokine quantification. FACS Analysis Cultured DC or single cell suspensions from PP or MLN were stained as described previously [12,61]. with different extracellular markers including CD11c, CD3, CD4, CD25 (BD Pharmingen, San Diego, CA) and intracellular IL-10, Foxp3 (eBiosciences, San Diego, CA), and HO-1 (Abcam, Cambridge, MA). For in- tracellular staining, the cells were initially stained for surface markers and then fixed, permeabilized and stained for intracellular expression of the markers as recommended by the related manufacturer. For intracellular cytokine expression, cells (16106 cells/ml) were stimulated by plate bonded anti-CD3 and soluble anti-CD28 in 96-well cell culture plates and were incubated for 6 h in the presence of the protein transport inhibitor GolgiStopTM (BD Biosciences) prior to the staining [61]. Data were acquired with LSRII or FACSCanto (Becton Dickinson, Oakville, Canada) and analyzed with the FlowJo program (TreeStar, Ashland, OR). Absolute numbers of cells were calculated by multiplying the percentage of positive staining cells in the total acquired events by the total number of cells isolated from the tissue analyzed. Cytometric Bead Array Assay 96-well cell culture plates were coated with anti-CD3 (10 mg/ ml) overnight. Cells were then plated at a density of 16106 cells/ ml with soluble anti-CD28 (2 mg/ml) for 48 h. Supernatants were collected and stored at 220uC prior to cytokine quantification. Cytokines in supernatants were analyzed with the mouse flex set cytokine cytometric bead array kit (Becton Dickinson, Canada) and BD FACSArray bioanalyzer. Statistical Analysis Experimental results are expressed as means 6 the standard errors of the means. Statistical analyses were performed by means of one-way analysis of variance (ANOVA), followed by Tukeys test for comparing all pairs of groups. Significant differences between two groups were determined using the unpaired Student’s t test. A statistical software package (GraphPad PRISMTM version 5.0) was used for the analysis. A p value of less than 0.05 was considered statistically significant. Author Contributions Conceived and designed the experiments: KK PF. Performed the experiments: KK NK JC PF. Analyzed the data: KK PF. Contributed reagents/materials/analysis tools: JB. Wrote the paper: PF KK JB. 6. Reid G, Hammond JA (2005) Probiotics. Some evidence of their effectiveness. Can Fam Physician 51: 1487–1493. 7. Forsythe P, Bienenstock J (2010) Immunomodulation by commensal and probiotic bacteria. Immunol Invest 39(4–5): 429–448. 8. Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, et al. (2005) Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 115(6): 1260–1267. 9. Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL (2005) An immunomod- ulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 122(1): 107–18. 10. Mazmanian SK, Round JL, Kasper DL (2008) A microbial symbiosis factor prevents intestinal inflammatory disease. Nature. 453(7195): 620–5. PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e47556