with bacteria are likely to be critical in orchestrating these immune responses. A range of mediators and enzymes expressed by DC, have been linked to the capacity of these cells to induce a tolerogenic or regulatory immune response including IL-10, TGFb, indoleamine 2,3 dioxygenase (IDO) and COX-2 [18–20]. Recently there has been increasing interest in the immunomod- ulatory actions of heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin [21]. Antigen presenting cells (APC) expressing HO-1 promote the formation and function of Foxp3+ Treg [22] and the enzyme also plays a critical role in active immune suppression by DC [23]. Here we investigate immune responses in the GALT following oral treatment with a strain of lactobacillus, L. rhamnousus JB-1, previously demonstrated to have immunomodulatory actions in a number of model systems [12,24,25]. Further, we address the potential role for DC and HO-1 in mediating the changes induced by this organism. Results Lactobacillus Rhamnosus JB-1, but not L.salivarius, Increases Foxp3+ T cell Population in Peyer’s Patches and Mesenteric Lymph Nodes Feeding with JB-1 lead to a significant increase in the percentage of CD4+ T cells expressing of Foxp3 in both the Peyer’s Patches (PP) and mesenteric lymph nodes (MLN). This increase was greatest in the MLN, where following 3 days of treatment the percentage of Foxp3+ CD4+ T cells increased from 1.960.05% to 5.660.1% and further increased to 15.660.25% and 14.260.1% at 5 and 9 days treatment respectively (Figure 1A,B&C) this reflected an increase in total number of Foxp3+ CD4+ cells from 15.960.26104 to 21364.6104 following 9 days treatment. While the Foxp3+ cell population also increased in the Peyer’s Patches this reached a statistically significant level only after 9 days of treatment (3.360.05% vs 9.061.0%, n = 5 p,0.05) with total Foxp3+ CD4+ cell numbers increasing from 0.3760.0036104 to 1.560.096104). When focusing on the expression of Foxp3 in the CD4+CD25+ population there was a small, statistically significant, increase in PP but again changes were most marked in the MLN (Figure 1D&E). The increase of CD4+CD25+Foxp3+ cells at both sites was observed following 3 days treatment (34.260.8% vs 41.761.2% and 25.360.5% vs 81.666.5% in PP and MLN respectively), these enhanced levels were maintained but did not increase further with up to 9 days of treatment (figure 1 D&E). We did not observe significant changes in Foxp3 expression within the CD4+CD25– population in either the PP or MLN (data not shown). Another strain of Lactobacillus, L.salivarius, that we had previously demonstrated to lack some of the immunomodulatory effects of JB-1 [12,24] did not induce increases in Foxp3 expression following up to 9 days treatment (figure 1), again suggesting that the observed effects on Foxp3 expression are dependent on strain of bacteria used. Lactobacillus Rhamnosus JB-1 Enhances the Suppressive Function of CD4+CD25+ Cells and Decreases Inflammatory Cytokine Production by T cells in the Mesenteric Lymph Nodes Having determined that there was an increase in Foxp3 expression in CD4+ CD25+ cells in the MLN of mice treated with JB-1 we wanted to assess whether this was associated with enhanced suppressor activity indicative of functional T regulatory cells. CD4+CD25+ cells from the MLN of JB-1 or vehicle-fed mice Lactobacillus Induced Heme Oxygenase 1 were freshly isolated ex vivo. These T regulatory cell subsets were co-cultured with CFSE-labeled CD4+ CD25– T cells from DO11.10 transgenic mice under activating conditions. Cells from JB-1 treated animals showed a significant (2 fold) increase in their ability to suppress proliferation of activated CD4+CD25– cells when compared to vehicle treated controls (Figure 2). The increase in Foxp3+ cells in the MLN was also associated with a corresponding decrease in TNF and IFNc release following stimulation of isolated MLN cells while IL-10 levels were not significantly altered (Figure 3A). The decrease in TNF and IFNc was significant following 3 days treatment and maximal at 5 days, corresponding with maximal Foxp3 expression. FACS analysis reveled that TNF production by CD4+ cells was decreased significantly after 3 days treatment and almost abolished at 5 days (Figure 3B). These data suggest the establishment of an anti- inflammatory/tolerogenic environment in the MLN following feeding with JB-1. Lactobacillus Exposure Induces Regulatory Dendritic Cells Expressing Heme Oxygenase Given the evidence that HO-1 can be an important functional component of regulatory DC [23,26] we investigated the ability of JB-1 to induce HO-1 expression in DC both in vitro and in vivo. Treatment of mice with JB-1 for 5 days lead to a significant increase in CD11c+ cells expressing HO-1 in the MLN (8.961.4 vs 17.862.2, n = 5 p,0.05) (Figure 4A) and while there was also an increase in HO-1 expression in DC in PP this was not statistically significant (4.661.1 vs 7.160.6, n = 4 p = 0.1). To determine if JB-1 must be alive to lead to the induction HO-1 we also treated with c-irradiated bacteria. The killed organisms failed to increase HO-1 in DC within the MLN. The increase in HO-1 could be mimicked by direct co-culture of bone marrow derived DC (BMDC) with JB-1 for 24 hrs (Figure 4B). As with Foxp3 expression in T cells induction of HO-1 was also strain specific as exposure to lactobacillus salivarius, in vivo or in vitro, did not significantly change HO-1 expression in DC (Figure 4). In striking contrast to our in vivo findings, killed JB-1 was even more effective in inducing HO-1 expression in DC, following in vitro exposure, than the live organism (Figure 4B). FACS analysis following in vitro exposure of DC to PKH26 labeled JB-1 indicated that 36.461.7% of the DC are associated with JB-1 after 24 h. Within the population of JB-1 associated DC, 61.960.6% express HO-1 compared with only 3.960.2% of DC not associated with JB-1 (Figure 5) suggesting that direct JB-1/DC interaction is required for the induction of HO-1. To determine whether DC and JB-1 interact in vivo we treated mice with CFSE labeled bacteria. Examination of Peyer’s patches 18 hours following gavage of bacteria revealed a population of CFSE bright DC (11.963.5% of CD11c+ cells) which was not observed in vehicle treated mice or mice treated with unlabeled JB-1. This suggests that JB-1 becomes associated with DC in the Peyer’s patches following feeding. As a test of immunoregulatory capacity, BMDC exposed to JB-1 in vitro were adoptively transferred into the footpad of mice. This lead to a significant increase in CD4+ Foxp3+ cells in the popliteal lymph node, while transfer of the same number of JB-1 alone, unexposed DC or DC exposed to L. salivarius had no significant effect on Foxp3 (Figure 6). Inhibition of Hemeoxygenase Prevents the Lactobacillus Induced Increase in Foxp3 Expression There is good evidence that certain immunoregulatory actions of HO-1+ DC are dependent on activity of the enzyme [27,28]. Therefore we utilized chromium mesoporphyrin (CrMP), a well- PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47556