Lactobacillus Induced Heme Oxygenase 1 Figure 4. Hemoxygenase-1 expression is up-regulated in dendritic cells following exposure to JB-1. Balb/c mice were given live or killed (gamma-irradiated) L.Rhamnosus JB-1, L.Salivarius (LS) or vehicle by gavage for 5 days. Single cell suspensions of mesenteric lymph nodes were stained for CD11c, MHCII, and intracellular HO-1 expression using flow cytometry. Shown are representative dot plots and corresponding graphs represents HO-1 (A) expression in mesenteric lymph node DC. (B) Dendritic cells were cultured from bone marrow and CD11c+ cells were purified and cocultured with JB-1 for 24 hr. HO-1 expression in DC was assessed by FACS. Data presented as mean 6 SEM n = 5 *P,0.05. doi:10.1371/journal.pone.0047556.g004 presenting cells has a more important role in immunosuppression [36,37]. Overexpression of HO-1 in DC leads to increased secretion of IL-10 and reduced capacity of the cells to trigger effector immune responses [38]. Recently, Mougiakakos et al [39] identified HO-1 expression in human mesenchymal stem cells and demonstrated that pharmacological inhibition of HO-1 activity lead to a significant reduction of the ability of these cells to induce regulatory T cells. George et al. identified that HO-1 expression by antigen presenting cells is required for the function of CD4+CD25+ Treg [22]. DC expressing HO-1 can also directly inhibit T cell proliferation and are involved in tolerance in a rat model of allotransplantation [23]. Similarly it has been reported that hepatic overexpression of HO-1 leads to induction of regulatory T cells that are responsible for increased survival of liver allografts [40]. Here we demonstrate that feeding with JB-1 leads to increased HO-1 expression by DC in the MLN and this effect was mimicked by direct in vitro exposure of bone marrow derived DC to the bacteria. This change in phenotype was associated with increased regulatory activity of these DC as evidenced by the ability to induce Foxp3 expression in lymph nodes following adoptive transfer. Live JB-1 was required to increase HO-1 expression in DC following oral treatment. This finding supports our previous studies showing that killed JB-1 lacked the Treg associated anti-inflammatory action of the live organism in a mouse model of asthma [12,24]. However, in vitro exposure of killed JB-1 to DC lead to an even greater induction of HO-1 than the live organism. The reasons behind this seeming paradox of in vitro and in vivo responses to the killed bacteria are unclear. However, it is likely that transit through the GI tract is very different for dead and live bacteria and that killed organisms may not reach DC in the Peyer’s patches in significant numbers, if at all. Unfortunately, we were unable to develop an effective method of labelling dead organisms to test this hypothesis within the current study. Similarly, we can currently only speculate on mechanisms underlying the enhanced response of DC to killed versus live bacteria in vitro. It may be that the expression of ligands for pattern recognitions receptors (PRR), such as Toll-like receptors (TLR) and NOD-like receptors (NLR) are altered in the killed bacteria. Alternatively, the c-irradiation may lead to small changes in the motifs recognized by PRR; it is known that even minor chemical modifications in bacterial cell wall components can result in significantly different immunological consequences [41,42]. Microbes can also interact with the host immune system by means of their genomic DNA; most notably unmethylated CpG motifs can activate immune responses in vitro and in vivo through activation of TLR9 [43]. DNA is the principal cellular target governing loss of viability after exposure to gamma-irradiation; with cell death predominantly induced by double-strand breaks in DNA that cannot be repaired by the cell [44]. It is possible that such changes in DNA influence immune effects of the bacteria. Clearly further investigations of the differences in the capacity of PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47556