Lactobacillus Induced Heme Oxygenase 1 Figure 8. Blocking IL-10 or TGFb does not prevent the suppression of T cell inflammatory cytokine production by JB-1 exposed DC. Isolated mesenteric CD11c+ DC were co-cultured with L. rhamnosus JB-1 at a ratio of 10:1/bacteria:DC, for 2 h at 37uC and incubated with purified CD4+CD25– responder T cells from DO11.10 transgenic mice for 5 days. T cells were harvested and stimulated by antibodies against CD3/CD28 in the presence of OVA protein for 48 hr and the supernatants analyzed for cytokine production. Where indicated, anti-IL-10 (1 mg/ml) or anti-TGFb (50 mg/ ml) was added to the co-culture. Data is presented as mean values 6 SEM n = 5, *p,0.05 compared to control values. doi:10.1371/journal.pone.0047556.g008 University (approval number 09–05–17). Adult male Balb/c and DO.11.10 transgenic mice (20–25 g) were maintained in an automatic light/dark cycle (light periods of 12 h) and provided water and chow ad libitum. Mice were acclimatized to the animal facility for 1 week before experimentation. Age-matched (8–9 wk old) animals were used in all experiments. Bacterial Treatment Two bacterial strains were used in this study, L. rhamnosus (JB-1), is the same as that employed in several published investigations [12,24,25] and was previously referred to as L reuteri. This strain was recently confirmed as a Lactobacillus rhamnosus, by AFLP fingerprinting and full genomic analysis. It was identified as a strain distinct from L rhamnosus GG [56], and any of the 118 L rhamnosus strains, examined by Vancanneyt et al. [57] and does not belong to any of the 7 clusters identified by the Bacteria Collection Laboratory for Microbiology, University of Ghent, Belgium. L. salivarius UCC118 were a gift from Dr. B. Kiely (Alimentary Health, Cork, Ireland). Both strains were prepared from frozen stocks (–80uC) as described previously [24]. Briefly, bacteria were suspended in Man-Rogosa-Sharpe liquid medium (MRS broth; Difco Laboratories, Detroit, MI) plated in MRS agar, cultured anaerobically at 37uC for 24 hours, then inoculated in fresh MRS broth and grown at 37uC under anaerobic conditions for 48 hours. Bacteria were washed twice with sterile phosphate-buffered saline (PBS) to a concentration of 66108 bacteria/ml as determined by a Vitek colorimeter (bioMe´rieux, Hazelwood, MO). Bacterial suspensions were then centrifuged and resuspended in MRS broth to give a concentration of 56109/ml24. Suspensions of L. rhamnosus JB-1 were killed by c-irradiation with Cobalt 60 for 20 hours at 8.05 Gy/minute. The resulting viability was determined by plating on MRS agar plates under anaerobic conditions for 72 hours at 37uC. No bacterial growth was detected in irradiated preparations after 72 hours of culture. Na¨ıve mice received 16109 JB-1 or L.salivarius in 200 ml of MRS liquid medium broth via oral gavage needle daily for 3, 5 or 9 days. Control animals were treated daily with 200 ml of MRS broth alone. Following treatments animals were euthanized and Peyer’s Patches and MLN removed and processed for FACS analysis or cell isolation. Inhibition of Heme oxygenase Heme oxygenase activity was inhibited in vivo using the potent and specific heme oxygenase inhibitor [30] Chromium(III) Mesoporphyrin IX chloride(CrMP) (Frontier Scientific Inc.). 25 mg/kg of CrMP was administered (i.p) daily over a period of 7 days starting 2 days before mice were gavaged with bacteria or MRS broth. Labeling of Bacteria Red fluorochrome PKH26 (Sigma-Aldrich, Okville, Canda) was used to label bacteria according to the manufacturer’s instructions. Briefly, 26108 bacteria were washed with PBS and the cell pellet was dissolved in PKH-26 diluent. PKH-26 fluorochrome was diluted in a separate tube in PKH-26 diluent. The bacterial suspension was added to the PKH-26-containing tube and incubated for 7 min at room temp. Staining was stopped by adding PBS containing fetal calf serum for 2 min. Bacteria were then washed three times with PBS and resuspended to the desired concentration in serum- and antibiotic-free medium prior to incubation with DC. For in vivo studies bacteria were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE) as described previously [58], with some modifications. Briefly, 56109 bacteria were washed with PBS and CFSE was added to the tube at final concentration of 5 mM and incubated for 10 min under constant shaking at room temperature. Staining was stopped by adding PBS containing FCS for 2 min. Bacteria were then washed three times with PBS, 5% FCS and resuspended in PBS at 56109/ml prior to gavage. Culturing BMDC Bone marrow cells were cultured in the presence of granulocyte monocyte colony stimulating factor for 7 days as previously described [59,60]. The cells were fed with fresh medium and cytokine on days 3 and 6 of the culture. On day 7 of culture DC PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47556