Lactobacillus Induced Heme Oxygenase 1 Figure 6. Dendritic cells exposed to JB-1 can induce Foxp3 expression in CD4+ T cells. Numbers (A) and percentage (B) of Foxp3+ CD4+ T cells in the popliteal lymph node of mice 5 days following adoptive transfer of JB-1 loaded BMDC into the footpad (16106 cells). Transfer of unloaded BMDC served as a control. Data presented as mean 6 SEM, n = 5, *p,0.05. doi:10.1371/journal.pone.0047556.g006 that a mutant strain of L. plantarum (Dlt-) that incorporates less d- Alanine in cell wall teichoic acids had a markedly reduced ability to induce secretion of proinflammatory cytokines from peripheral blood mononuclear cells while stimulating increased IL-10 pro- duction. The observed increase in HO-1 expression occurred almost exclusively in those DC associated with JB-1 or components of the bacteria, suggesting that direct interaction of the bacteria and DC is required to mediate the phenotypic and functional changes and indeed we observed uptake of CFSE labelled JB-1 by DC in the Peyer’s patches demonstrating that such interaction occurs in vivo. The fact that we did not detect statistically significant increases in HO-1 expression by DC within the Peyer’s patches may suggest that, upon phagocytosis of JB-1, DC migrate quickly to the MLN and that while induction of HO-1 is triggered within the Peyer’s patches it develops over time as the DC migrate. The DC receptors that might be involved in mediating these effects are currently unknown. Smits et al [8] demonstrated that certain lactobacilli that induced T cells to produce IL-10 when cultured with human monocyte-derived DC bound the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non- integrin (DC-SIGN). Blocking antibodies to DC-SIGN inhibited the induction of the Treg cells by these lactobacilli, indicating that ligation of DC-SIGN can actively prime DC to induce Treg cells. However there is good evidence that additional receptors in- cluding toll-like receptors 2,4 and 9 are critical to the tolerogenic and/or anti-inflammatory effects of probiotics [24,33,43,47,48] and it is likely that multiple receptors are involved in determining differential immunological responses following exposure to specific bacterial strains. It has been demonstrated that inhibitors of TGFb, COX-2 or IDO can prevent DC induced Foxp3 expression in the CD4+CD25– cell population following treatment with a mixed strain probiotic preparation, suggesting that multiple components of regulatory DC are required to influence Treg generation [18]. Through use of the heme oxygenase inhibitor, chromium mesoporphyrin, we have now identified heme oxygenase as playing an important role in induction of CD4+CD25+Foxp3+ Treg. Whether differential expression of regulatory mediators in DC determines bacterial strain specific induction of Treg phenotypes remains to be determined. Interestingly, while inhibition of heme oxygenase prevented the increase in Foxp3 expression by CD4+CD25+ cells, in vivo and in vitro, it did not alter the inhibition of TNF and IFNc production by T cells induced by JB-1. In addition the presence of either anti- IL10 or anti-TGFb did not modulate this inhibition of in- flammatory cytokine production. These results suggest that the increase in Foxp3 expressing Treg is independent of the associated decrease in inflammatory cytokine production and that while HO- 1 represents a component of the immunoregulatory pathway leading to the induction of Foxp3+ expression it does not influence effector T cell activity in the MLN. Thus it appears that JB-1 feeding triggers multiple regulatory pathways. The nature of these additional regulatory pathways remains to be determined but could include direct suppressive action of DC through COX-2 or IDO mediated responses [18] or the induction of a Foxp3- population of regulatory T cells. Our findings indicating role for HO-1 in Foxp3 induction but not in suppression of inflammatory cytokine production by CD4+ T cells contrasts those of Moreau et al [23] which suggested that, in vitro, HO-1 activity of DC does not play a role in the stimulation and expansion of Treg in vitro but instead directly inhibits T cell proliferation. However there are a number of in vivo studies that clearly support a role for HO-1 in promoting Foxp3+ Treg, [28,47,48] for example Xia et al showed that HO-1 activity is required for up-regulation of Foxp3+ Treg and subsequent attenuation of ovalbumin induced airway inflamma- tion induced by the heme oxygenase activator, hemin [49]. It may be that in vivo, expression of HO-1 by other cells types is involved in promoting Foxp3 expression but it is clear from our adoptive transfer experiments that JB-1 exposed DC do have the ability to induce Foxp3 expression. PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47556