Figure 1.
Dexamethasone modulates cytokine cocktail-induced DC maturation.
(A) Phenotypic analysis of untreated (iDCs), cytokine-activated (mDCs) and 10−6 M dexamethasone cytokine-activated dendritic cells (Tol-DCs) was performed by flow cytometry. Representative histogram data set from 12 independent experiments is shown. Maturation associated molecules are depicted in the lower graph as mean fluorescent intensity of expression (MFI) of mDCs and Tol-DCs relative (fold-change expression) to iDCs. (B) IL-10 and IL-12p70 were measured in supernatants harvested from DCs. Concentration of IL-10 (in pg/ml) is shown (n = 15). In none of the conditions analyzed were IL-12p70 or IL-23 produced (lowest detection limit 7.6 pg/ml). (C) Transcripts levels of IL-10 and IL-12p35 were determined by real-time PCR using β-actin as the endogenous reference gene. Data represent fold-change induction relative to iDCs (n = 3). Student’s t-test: *p<0.05, **p<0.001.
Figure 2.
Tol-DCs have a reduced capacity to stimulate T lymphocytes.
(A) DCs were cultured with allogeneic PBL at different ratio (1∶20 or 1∶100) for seven days. Upper-left panel data represent the mean ± SD of a representative experiment carried out in triplicate of the seven (upper-right graph) that were independently performed. (B) Antigen-specific T-cell responses. CD4+ T cells we cultured with autologous DCs pre-loaded with the superantigen TSST-1 (left graph) or with tetanus toxoid (+ presence and – absence of TT) at a 1∶20 ratio for seven days. T-cell proliferation was determined in triplicate by 3H thymidine incorporation. Data represent the mean ± SD of n = 3 independently performed experiments. Student’s t-test: *p<0.05, **p<0.001.
Figure 3.
Tol-DCs induce anergic T cells.
(A) Naïve CD4+ CD45RA++ T cells were primarily primed with allogeneic iDCs, mDCs or tol-DCs for 7 days. After 5 days, anergy induction was examined by re-stimulation of primed CD4+ T cells with iDCs or mDCs from the original donor. (B) TT-specific CD4+ T cells were primed with TT-loaded autologous iDCs, mDCs or tol-DCs for 6 days (initial challenge). After in vitro expansion with TT loaded-DCs anergy induction was examined by re-stimulation of TT-specific CD4+ T cells with mDCs loaded (+) with TT at a 1∶20 ratio. Data represent the mean ± SD of n = 5 experiments that were independently performed. Proliferation was normalized relative to mDCs loaded with TT (100%) for each independent experiment. Cytokines were determined in the supernatant of cell cultures by ELISA (<d; below detection limit; IFN-γ data represent mean ± SD of n = 3).
Figure 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 µg/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-γ and IL-10 production in the supernatant was analyzed.
Figure 5.
Gram-negative bacteria do not break 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-α (40%; p<0.05) and elevation of IL-10 (78% increase; p<0.05) in Gram-negative treated DCs. (C) Gram-negative stimulated DCs were cultured after being carefully washed with allogenic PBLs (ratio 1∶20) for 7 days. The % of proliferating cells was measured by CFSE dilution using flow cytometry. Significant allo-response inhibition of E. coli dex-DC (inhibition 28%; p<0.05) compared to control DCs. IFN-γ secretion was analyzed in the supernatant by standard ELISA. Results represent the mean and standard deviation of three independent donors. Student’s t-test: *p<0.05, **p<0.001.
Figure 6.
Gram negative E. coli induces tolerogenic activation on Tol-DCs.
DCs were carefully washed to eliminate cytokines and dexamethasone at day 7, and viable DCs were further re-challenged with E. coli (ratio 1∶10) without cytokines or dexamethasone. (A) Tol-DCs (dex matured-DCs) produced significant higher levels of IL-10 whereas levels of pro-inflammatory cytokines were very low compared with mDCs or iDCs in response to E. coli (n = 4, from each donor, iDCs, mDCs and tol-DCs were generated in parallel). (B) The production of IFN-γ was evaluated in the supernatant of allogenic T cells cultured for 7 days with E. coli stimulated mDCs or tol-DCs. IFN-γ production was significantly (p = 0.024) reduced in T cells stimulated with tol-DCs plus E. coli. IL-10 was not detected in any condition (data not included). Student’s t-test: *p<0.05, **p<0.001.
Figure 7.
Tol-DCs interaction with Gram-negative enterobacteria inhibits Th1 response.
Tol-DCs were treated as described in figure 5 and 6. Proliferative response and IFN-γ production induced by Gram-negative enterobacteria (P. mirabillis, K. pneumoniae and S. thyphimurium) stimulation of dex-DCs (A) and tol-DCs (dex matured-DCs) (B) were evaluated in allogeneic T cell culture. IFN-γ production was reduced in T cells stimulated with tol-DCs plus Gram-negative enterobacteria. IL-10 was not detected. Data represent mean ± SD of four independent experiments. Student’s t-test: *p<0.05.
Figure 8.
Crohn’s disease patients’ DCs are educated towards tolerogenic phenotype.
(A) Maturation associated molecules upregulation in DCs from Crohn’s disease patients are depicted as mean fluorescent intensity of expression (MFI) in mDCs and tol-DCs relative to iDCs (fold-change expression). (B) IL-10 was measured in supernatants harvested from DCs. Concentration of IL-10 (in pg/ml) is shown as mean ± SD (n = 6). (C) Proliferative response and IFN-γ production induced by tol-DCs from patients were evaluated in allogeneic T cell culture. Both, proliferation and IFN-γ production were reduced in T cells stimulated with tol-DCs compared to mDCs (data represent mean± SD (n = 4)). IFN-γ production was normalized relative to mDCs (100%) for each independent experiment (n = 3). Student’s t-test: *p<0.05.