Western blot analysis HEK293 or other cells were cultured in 6-well plates

effective in inducing HO-1 expression in DC, following in vitro exposure, than the live organism. 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 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 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. 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 MedChemExpress c-Met inhibitor 2 enhanced suppressor activity indicative of functional T regulatory cells. CD4+CD25+ cells from the MLN of JB-1 or vehicle-fed mice Inhibition of Hemeoxygenase Prevents the Lactobacillus Induced Increase in Foxp3 Expression Lactobacillus Induced Heme Oxygenase 1 3 Lactobacillus Induced Heme Oxygenase 1 characterized HO-1 inhibitor to determine the role of this enzyme in JB-1 mediated changes in the MLN. Treatment of mice with CrMP markedly attenuated the increase in CD4+CD25+Foxp3+ cells induced by JB-1. These findings were mimicked in vitro. Isolated MLN DC exposed to JB1 before co-culture with CD4+ T cells induced an increase in the expression of Foxp3 in the CD4+CD25+ T cell population while addition of CrMP to the co-culture prevented this increase. between an ex vivo and an entirely in vitro system and the response of transgenic, OVA specific, T cells in contrast to MLN cells. However treatment with neither anti-IL-10 nor anti-TGFb prevented the JB-1 exposed DC induced reduction of IFN and TNF release by T cells. Overall these data suggest that HO-1 activity is an important component of Foxp3 induction following JB-1 feeding. However, it is clear that the bacteria trigger additional, HO-1, Foxp3, TGFb and IL-10 independent, regulatory pathways that contribute to the tolerogenic environment of the MLN. Inhibition of Heme oxygenase does not Prevent the Lactobacillus Induced Decrease in Inflammatory Cytokine Production While treatment of mice with CrMP attenuated the increase in CD4+CD25+Foxp3+ cells induced by JB-1, inhibition of HO-1 did not prevent the decrease in IFNc and TNF production in the MLN. To further investigate the mechanism underlying this decrease in IFNc and TNF production we utilized an in vitro system, coculturing JB-1 exposed DC with CD4+CD252 responder T cells from DO11.10 transgenic mice and assessing the effect of blocking signaling of the major regulatory cytokines IL-10 or TGFb on T cell cytokine production. DC isolated from M