Figure 1.
Increase in Foxp3+ T regulatory cells in Peyer’s patches and mesenteric lymph nodes following JB-1 feeding.
Balb/c mice were given L. Rhamnosus JB-1, (LR) L.salivarius (LS) or vehicle by gavage for 3, 5 or 9 days. Single cell suspensions of Peyer’s patches (PP) or mesenteric lymph nodes (MLN) stained for CD3, CD4 and Foxp3 and analyzed by flow cytometry. Representative dot plots (A) and mean ± SEM values of Foxp3+ cells as a percentage of total (B&C) and absolute number (D&E) among the CD3+CD4+ cells and absolute number among the CD3+ CD4+ CD25+ (F&G) on days 3, 5, and 9 in lactobacillus and vehicle-fed mice are depicted. n = 5 *p<0.05.
Figure 2.
JB-1 enhances the in vitro suppressive capacity of MLN CD4+CD25+ cells.
(A) CFSE plots, representative from 4 similar experiments, of the T effector–cell proliferation in the absence (tinted histograms), and presence (open histogram), of T regulatory cells at a Treg to T effector cell ratio of 1∶8 (B) Percentage of inhibition of effector T-cell proliferation by T regulatory cells from treated (JB-1) and vehicle-fed controls. Data presented as mean ± SEM n = 4 *P<0.05. CFSE = carboxyfluorescein diacetate succinimidyl ester.
Figure 3.
Feeding of JB-1 reduces secretion of inflammatory cytokines by T cells.
Balb/c mice were given L. rhamnosus (JB-1), L.salivarius (LS) or vehicle by gavage for 3 or 5 days. (A) Single cell suspensions of mesenteric lymph nodes were stimulated by antibodies against CD3/CD28 for 48 hr and the supernatants were analyzed for cytokine productions using Cytometric Bead Array Assay. Mean ± SEM values for TNF, IFN-γ and IL-10 release are depicted (n = 6 a, b = P<0.05 compared to vehicle and LS respectively). (B) Single cell suspensions of mesenteric lymph nodes were stimulated by PMA and ionomycin for 4 hr and intracellular TNF by CD4+ CD3+ T cells were depicted data are representative of three similar independent experiments.
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 ± SEM n = 5 *P<0.05.
Figure 5.
HO-1 expression is enhanced in Dendritic cells assocaited with JB-1.
Dendritic cells were cultured from bone marrow and CD11c+ cells isolated and cocultured with PKH26 labeled L.rhamnosus JB-1 for 24 hr and HO-1 expression assessed by FACS. A) A dot plot showing a subpopulation of dendritic cells associated with the labeled lactobacillus. This is representative of 4 similar experiments. B) Representative dot plots and C) mean values of the percentage HO-1+ cells in the Lactobacillus associated (JB-1+ve) and non-associated (JB-1-ve) dendritic cell populations. Evidence for in vivo interaction between DC and JB-1 is provided by D) Representative dot plots and E) mean values of CFSE positive cells within the CD11c positive population from Peyers Patches of mice fed with CFSE labeled or unlabeled JB-1. Data presented as mean ± SEM, n = 5, *p<0.05. CFSE = carboxyfluorescein diacetate succinimidyl ester.
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 (1×106 cells). Transfer of unloaded BMDC served as a control. Data presented as mean ± SEM, n = 5, *p<0.05.
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 37°C and incubated in the presence or absence of CrMP (5 µM) 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 IFNγ, relative to vehicle control (TNF 398±49.2 pg/ml, IFNγ 592±105 pg/ml) is depicted. Data is presented as mean values ± SEM n = 5, *p<0.05 compared to control values.
Figure 8.
Blocking IL-10 or TGFβ does not prevent the suppression of T cell inflammatory cytokine production by JB-1 exposed DC.
Isolated mesenteric CD11c+ DC were co-cultured with L. rhamnosus JB-1 at a ratio of 10∶1/bacteria:DC, for 2 h at 37°C and incubated with purified CD4+CD25– responder T cells from DO11.10 transgenic mice for 5 days. T cells were harvested and stimulated by antibodies against CD3/CD28 in the presence of OVA protein for 48 hr and the supernatants analyzed for cytokine production. Where indicated, anti-IL-10 (1 µg/ml) or anti-TGFβ (50 µg/ml) was added to the co-culture. Data is presented as mean values ± SEM n = 5, *p<0.05 compared to control values.