Lactobacillus Induced Heme Oxygenase 1 Figure 3. Feeding of JB-1 reduces secretion of inflammatory cytokines by T cells. Balb/c mice were given L. rhamnosus (JB-1), L.salivarius (LS) or vehicle by gavage for 3 or 5 days. (A) Single cell suspensions of mesenteric lymph nodes were stimulated by antibodies against CD3/CD28 for 48 hr and the supernatants were analyzed for cytokine productions using Cytometric Bead Array Assay. Mean 6 SEM values for TNF, IFN-c and IL-10 release are depicted (n = 6 a, b = P,0.05 compared to vehicle and LS respectively). (B) Single cell suspensions of mesenteric lymph nodes were stimulated by PMA and ionomycin for 4 hr and intracellular TNF by CD4+ CD3+ T cells were depicted data are representative of three similar independent experiments. doi:10.1371/journal.pone.0047556.g003 by studies demonstrating that Foxp3+ T cell generated in the MLN following exposure to a mixed probiotic population can migrate to sites of inflammation in experimental atopic dermatitis [18]. Here we further investigated the immunomodulatory effects of JB-1. We demonstrated that oral treatment with this bacteria lead to a significant increase in CD4+CD25+Foxp3+ cells in the PP and MLN. The increase in these cells was most marked in the MLN, this is not surprising given its central and critical roles in induction of tolerance/immunity and gating of communication between the gut mucosa and systemic immune system [31]. Interestingly, studies colonizing germ free mice with clostridia [3] or altered Schaedler flora [4] demonstrated regulatory T cell induction in the colonic lamina propria but not in the Peyer’s patches or mesenteric lymph node suggesting immunomodulatory pathways distinct from those observed with JB-1. The increase in CD4+CD25+Foxp3+ cells is accompanied by a decrease in CD3/CD28 induced TNF and IFNc production by T cells, with the maximal Foxp3 induction and proinflammatory cytokine inhibition occurring following 5 days treatment. Thus, oral treatment with JB-1 leads to the development of character- istics of a tolerogenic environment in the MLN. Oral tolerance is a key feature of intestinal immunity and generates systemic tolerance to fed antigens. The systemic aspects of tolerance are believed to be mediated by Treg however antigen/DC interaction and the development of regulatory DC phenotype in the MLN is critical to instigating tolerogenesis. DC are pivotal in early bacterial recognition [17,32]. Mac- pherson and Uhr [15] demonstrated that intestinal DC retain small numbers of live commensals for several days, while in vitro, mouse BMDC can take up various lactobacilli by phagocytosis which subsequently influences the ability of the BMDC to produce cytokines [33]. However, few studies have directly linked changes in DC phenotype with Treg induction by commensal bacteria. Kwon et al [18] reported that administration of a mixture of 5 probiotic strains induced generation of CD4+Foxp3+ Tregs from the CD4+CD25– population. This Treg induction was directly mediated by regulatory DC expressing high levels of IL-10, TGF- b, COX-2, and IDO. Our data supports a similar sequence of events, but demonstrates that a single bacterial strain can induce regulatory DC, primarily in the MLN, that can in turn drive the formation of CD4+CD25+ Foxp3+ cells. In direct contrast to Kwon et al [18] we identified an increase in Foxp3 expression in the CD25+ but not CD25– T cells. One possibility is that there is bacterial strain specific activation of distinct Treg populations. Crucially we identified increased HO-1 expression in DC following treatment with JB-1. Heme oxygenase is an enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin carbon monoxide and iron [21]. It has been identified that the inducible isoform, HO-1, is involved in immune regulation [21]. While there have been studies suggesting that HO-1 expression by regulatory T cells plays a critical role in their suppressive activity [34,35] more recent investigations have disputed this and instead evidence suggests that HO-1 by antigen PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47556