Lactobacillus Induced Heme Oxygenase 1 Figure 7. Heme oxygenase-inhibition attenuates Lactobacillus induced foxp3 expression in mesenteric lymph nodes but does not interfere with down-regulation of T cell inflammatory cytokine release. Balb/c mice received i.p. Chromium mesoporpyrin (CrMP) daily for 7 days. Mice were gavaged with JB-1 or vehicle for the last 5 days of experiment and mesenteric lymph nodes were harvested. (A) Single cell suspensions of mesenteric lymph nodes stained for CD4, CD25 and Foxp3 and analyzed by FACS. (B) Isolated mesenteric CD11c+ DC were co- cultured with L. rhamnosus JB-1 at a ratio of 10:1/bacteria:DC, for 2 h at 37uC and incubated in the presence or absence of CrMP (5 mM) with purified CD4+ T cells from DO11.10 transgenic mice for 5 days stained for CD4, CD25 and Foxp3 and analyzed by FACS. (C&D) Single cell suspensions of mesenteric lymph nodes were stimulated by antibodies against CD3/CD28 for 48 hr and the supernatants analyzed for cytokine production. Release of TNF and IFNc, relative to vehicle control (TNF 398649.2 pg/ml, IFNc 5926105 pg/ml) is depicted. Data is presented as mean values 6 SEM n = 5, *p,0.05 compared to control values. doi:10.1371/journal.pone.0047556.g007 In conclusion, we have demonstrated that oral treatment with JB-1 induces a tolerogenic environment in the MLN that includes the generation of HO-1+ regulatory DC. Furthermore, we have identified heme oxygenase activity as an important mediator of Foxp3+ Treg induction by this bacteria. However, additional, as yet unknown, regulatory pathways are involved in JB-1 mediated down-regulation of inflammatory cytokine production in the MLN. Given the extant literature suggesting a protective effect of increased HO-1 expression in models of asthma and in- flammatory bowel disease [28,48,50–55], disorders that are also suggested targets of microbial based or probiotic therapies, it will be interesting to determine whether activation of this enzyme is an important element of beneficial effects described for certain commensal organisms in these systems. Materials and Methods Animals These experiments were performed in strict accordance with guidelines of the Canadian Council for Animal Care. Protocols were approved by the Animal Research Ethics Board of McMaster PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47556