Lls exposed to tol-DCs from Crohn’s disease patients exhibited a significantly reduced capacity to proliferate (mean cpm = 20561613058 vs 38181618177; p = 0.037) compared to mDCs, as well as reduced IFN-c secretion when cocultured with fully competent mDCs (figure 8C). These results show the ability to generate tol-DCs in patients with Crohn’s disease.DiscussionThe generation of reproducible and stable clinical-grade tolerogenic DCs is a critical step towards developing therapeutic trials for the treatment of human disorders such as allergies, autoimmune diseases, chronic inflammation, and transplant rejection [19] [24]. The addition of immunosuppressive agents,pharmacological modulation, or inhibitory cytokines when DCs are being generated from monocytes influences the functional properties of the resulting DCs [9,10]. Several agents, including glucocorticoids [25] such as dexamethasone [26,27], mycophenolic acid [28], vitamin D3 (1a,25-dyhydroxyvitamin D3) [29], retinoic acid [30], the combination of dexamethasone and vitamin D3 [31], or IL-10 [32] have been used to render DCs resistant to maturation [33]. Tolerogenic DCs have been shown to induce T-cell anergy [34], suppress effector T cells, and promote the generation of regulatory T cells (Tregs) [14,35]. Interestingly, some studies [14] have reported that the maturation of dex-conditioned DCs with LPS potentiates the tolerogenic phenotype of DCs. We performed a detailed phenotype analysis in order to compare iDCs and fully mature DCs with tol-DCs from healthy donors and patients with Crohn’s disease and address the stability of tol-DCs. DCs conditioned with dexamethasone displayed a semi-mature phenotype, which is consistent with the tolerogenic DC phenotypes described elsewhere [36]. We also observed an alteration in the DC maturation process; 18325633 characterized by low-Tolerogenic 3PO Dendritic Cells Response to BacteriaFigure 4. Tol-DCs possess a stable phenotype. DCs were carefully washed to eliminate cytokines and dexamethasone, and viable DCs were further re-challenged with 100 ng/ml of LPS or 1 mg/ml of soluble CD40L as second stimuli. After 24 h, the phenotype (A) was analyzed by flow cytometry. Data represent relative MFI increase induced by LPS (n = 6) or CD40L (n = 4) compared to unstimulated iDCs, mDCs or tol-DCs as control. (B) IL-10 concentration is shown in pg/ml. IL-12p70 and IL-23 were not detected (detection limit = 7.8 pg/ml). Student’s t-test: *p,0.05, **p,0.001. (C) Tol-DCs do not recover the ability to stimulate T cells after re-challenge. T-cell proliferation was determined in triplicate by 3H-thymidine incorporation. IFN-c and IL-10 production in the supernatant was analyzed. doi:10.1371/journal.pone.0052456.gTolerogenic Dendritic Cells Response to BacteriaFigure 5. Gram-negative bacteria do not break 1527786 the tolerogenic properties of dexamethasone-DCs. Heat-killed bacteria were added at ratio 1:10 for 48 h to mo-DCs treated with dexamethasone or untreated as a positive control. A. Phenotypic analysis revealed statistically significant reduction of CD83, CD86, and MHC I and class II expression. Maturation associated molecules are depicted as mean fluorescent intensity of expression (MFI) of E. coli stimulated-DCs relative (fold-change expression) to BIBS39 cost control DCs without E. coli. (B) Cytokines produced by E. coli-stimulated DCs. Reduction of IL-12p70 (95.9 ; p,0.05), IL-23 (70.5 ; p,0.05) and TNF-a (40 ; p,0.05) and elevation of IL-10 (78 increase; p,0.05) in Gramnegative.Lls exposed to tol-DCs from Crohn’s disease patients exhibited a significantly reduced capacity to proliferate (mean cpm = 20561613058 vs 38181618177; p = 0.037) compared to mDCs, as well as reduced IFN-c secretion when cocultured with fully competent mDCs (figure 8C). These results show the ability to generate tol-DCs in patients with Crohn’s disease.DiscussionThe generation of reproducible and stable clinical-grade tolerogenic DCs is a critical step towards developing therapeutic trials for the treatment of human disorders such as allergies, autoimmune diseases, chronic inflammation, and transplant rejection [19] [24]. The addition of immunosuppressive agents,pharmacological modulation, or inhibitory cytokines when DCs are being generated from monocytes influences the functional properties of the resulting DCs [9,10]. Several agents, including glucocorticoids [25] such as dexamethasone [26,27], mycophenolic acid [28], vitamin D3 (1a,25-dyhydroxyvitamin D3) [29], retinoic acid [30], the combination of dexamethasone and vitamin D3 [31], or IL-10 [32] have been used to render DCs resistant to maturation [33]. Tolerogenic DCs have been shown to induce T-cell anergy [34], suppress effector T cells, and promote the generation of regulatory T cells (Tregs) [14,35]. Interestingly, some studies [14] have reported that the maturation of dex-conditioned DCs with LPS potentiates the tolerogenic phenotype of DCs. We performed a detailed phenotype analysis in order to compare iDCs and fully mature DCs with tol-DCs from healthy donors and patients with Crohn’s disease and address the stability of tol-DCs. DCs conditioned with dexamethasone displayed a semi-mature phenotype, which is consistent with the tolerogenic DC phenotypes described elsewhere [36]. We also observed an alteration in the DC maturation process; 18325633 characterized by low-Tolerogenic Dendritic Cells Response to BacteriaFigure 4. Tol-DCs possess a stable phenotype. DCs were carefully washed to eliminate cytokines and dexamethasone, and viable DCs were further re-challenged with 100 ng/ml of LPS or 1 mg/ml of soluble CD40L as second stimuli. After 24 h, the phenotype (A) was analyzed by flow cytometry. Data represent relative MFI increase induced by LPS (n = 6) or CD40L (n = 4) compared to unstimulated iDCs, mDCs or tol-DCs as control. (B) IL-10 concentration is shown in pg/ml. IL-12p70 and IL-23 were not detected (detection limit = 7.8 pg/ml). Student’s t-test: *p,0.05, **p,0.001. (C) Tol-DCs do not recover the ability to stimulate T cells after re-challenge. T-cell proliferation was determined in triplicate by 3H-thymidine incorporation. IFN-c and IL-10 production in the supernatant was analyzed. doi:10.1371/journal.pone.0052456.gTolerogenic Dendritic Cells Response to BacteriaFigure 5. Gram-negative bacteria do not break 1527786 the tolerogenic properties of dexamethasone-DCs. Heat-killed bacteria were added at ratio 1:10 for 48 h to mo-DCs treated with dexamethasone or untreated as a positive control. A. Phenotypic analysis revealed statistically significant reduction of CD83, CD86, and MHC I and class II expression. Maturation associated molecules are depicted as mean fluorescent intensity of expression (MFI) of E. coli stimulated-DCs relative (fold-change expression) to control DCs without E. coli. (B) Cytokines produced by E. coli-stimulated DCs. Reduction of IL-12p70 (95.9 ; p,0.05), IL-23 (70.5 ; p,0.05) and TNF-a (40 ; p,0.05) and elevation of IL-10 (78 increase; p,0.05) in Gramnegative.