Lactobacillus Induced Heme Oxygenase 1 Figure 1. Increase in Foxp3+ T regulatory cells in Peyer’s patches and mesenteric lymph nodes following JB-1 feeding. Balb/c mice were given L. Rhamnosus JB-1, (LR) L.salivarius (LS) or vehicle by gavage for 3, 5 or 9 days. Single cell suspensions of Peyer’s patches (PP) or mesenteric lymph nodes (MLN) stained for CD3, CD4 and Foxp3 and analyzed by flow cytometry. Representative dot plots (A) and mean 6 SEM values of Foxp3+ cells as a percentage of total (B&C) and absolute number (D&E) among the CD3+CD4+ cells and absolute number among the CD3+ CD4+ CD25+ (F&G) on days 3, 5, and 9 in lactobacillus and vehicle-fed mice are depicted. n = 5 *p,0.05. doi:10.1371/journal.pone.0047556.g001 characterized HO-1 inhibitor [29,30] to determine the role of this enzyme in JB-1 mediated changes in the MLN. Treatment of mice with CrMP markedly attenuated the increase in CD4+CD25+Foxp3+ cells induced by JB-1 (Figure 7A). These findings were mimicked in vitro. Isolated MLN DC exposed to JB- 1 before co-culture with CD4+ T cells induced an increase in the expression of Foxp3 in the CD4+CD25+ T cell population while addition of CrMP (5 mM) to the co-culture prevented this increase (Figure 7B). Inhibition of Heme oxygenase does not Prevent the Lactobacillus Induced Decrease in Inflammatory Cytokine Production While treatment of mice with CrMP attenuated the increase in CD4+CD25+Foxp3+ cells induced by JB-1, inhibition of HO-1 did not prevent the decrease in IFNc and TNF production in the MLN (Figure 7C&D). To further investigate the mechanism underlying this decrease in IFNc and TNF production we utilized an in vitro system, co- culturing JB-1 exposed DC with CD4+CD252 responder T cells from DO11.10 transgenic mice and assessing the effect of blocking signaling of the major regulatory cytokines IL-10 or TGFb on T cell cytokine production. DC isolated from MLN and exposed to JB-1 could mimic effects on T cells observed following feeding decreasing the release of IFNc and TNF following stimulation (Figure 8). While the decrease in cytokine production was significant the magnitude of the was less than observed in the isolated MLN cells following feeding of the bacteria (Figure 7 C&D) this likely reflects difference between an ex vivo and an entirely in vitro system and the response of transgenic, OVA specific, T cells in contrast to MLN cells. However treatment with neither anti-IL-10 nor anti-TGFb prevented the JB-1 exposed DC induced reduction of IFN and TNF release by T cells (Figure 8). Overall these data suggest that HO-1 activity is an important component of Foxp3 induction following JB-1 feeding. However, it is clear that the bacteria trigger additional, HO-1, Foxp3, TGFb and IL-10 independent, regulatory pathways that contribute to the tolerogenic environment of the MLN. Discussion Recent studies utilizing germ free mice have identified that key species within the microbiota, such as clostridia, single bacterial strains, or components of these strains, can strongly influence the development of T regulatory cells in the intestine [3–5]. In the present study we were attempting to gain further understanding of the immunological mechanisms underlying the ability of a single bacterial strain, previously demonstrated to have anti-inflamma- tory effects, to induce immunosuppression in an adult animal against the background of existing conventional microflora. It is suggested that the immunomodulatory effect of oral treatment with certain commensal organisms are associated with induction of Foxp3+ Treg and that these cells, induced in the GALT, can spread to sites beyond the intestine in response to immune challenge [12,13,18]. This concept is supported by our previous findings that oral treatment with JB-1 results in an increase in Treg in the draining lymph nodes of the lung of OVA sensitized mice following antigen challenge in the airway [12], and Figure 2. JB-1 enhances the in vitro suppressive capacity of MLN CD4+CD25+ cells. (A) CFSE plots, representative from 4 similar experiments, of the T effector–cell proliferation in the absence (tinted histograms), and presence (open histogram), of T regulatory cells at a Treg to T effector cell ratio of 1:8 (B) Percentage of inhibition of effector T-cell proliferation by T regulatory cells from treated (JB-1) and vehicle-fed controls. Data presented as mean 6 SEM n = 4 *P,0.05. CFSE = carboxyfluorescein diacetate succinimidyl ester. doi:10.1371/journal.pone.0047556.g002 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47556