Article Figures & Data
Figures
- FIG 1
DC cytotoxicity induced by bacterial EMs and silica NPs. (A) DCs were incubated for 24 h with different NP concentrations in the presence or absence of the indicated EMs. The supernatants were aspirated, and cells were incubated with MTS solution until color development. The percent MTS reduction was calculated with respect to the amount for nontreated cells (control). Data are the means ± SEs (n = 3, run in triplicate). *, P < 0.05 with respect to cells not treated with EMs. (B) IC50 (SiO2 NP doses resulting in 50% of the maximal cytotoxic effect, as measured by the MTS assay) values were plotted against EM concentrations. Data are means ± SEs (n = 3, run in triplicate). *, P < 0.05 with respect to cells treated with NPs alone. (C) DCs, treated as described above, were stained with annexin V-FITC and propidium iodide (PI) and subsequently subjected to FACS analysis. Bars represent the mean percentage ± SE (n = 3) for assays run in triplicate of PI-positive or annexin V-positive cells. *, P < 0.05 with respect to cells treated with NPs alone.
- FIG 2
Coincubation of DCs with SiO2 NPs and bacterium-derived EMs induces the release of IL-1β. DCs were incubated for 24 h with different concentrations of SiO2 NPs and the culture supernatants (EMs) from different Gram-negative bacteria (A and B) or Gram-positive bacteria (C and D) or E. coli LPS as a positive control (E). The level of extracellular IL-1β was measured by ELISAs. Data are the means ± SEs (n = 4, performed in triplicate). Black asterisks, P < 0.05 with respect to cells treated with NPs alone for all EM concentrations used; green or red asterisks, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green and red symbols, respectively).
- FIG 3
Expression profile of cytokines induced by coincubation of DCs with SiO2 NPs and bacterium-derived EMs. The extracellular media from DCs that had been treated for 24 h with SiO2 NPs and EMs, as indicated, were collected, and the concentrations of the indicated cytokines were measured by ELISAs. Data are the means ± SEs (n = 4, performed in triplicate). Black asterisks, P < 0.05 with respect to cells treated with NPs alone for all EM concentrations used; green or red asterisks, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green and red symbols, respectively). N.D., not determined. Orange straight lines, IC50 values; red boxes, the induction of cytokine release occurred at concentrations less than the IC50 values.
- FIG 4
Modulation of the expression of activation markers on DCs coincubated with SiO2 NPs and bacterium-derived EMs. DCs were incubated for 24 h with SiO2 NPs alone or in the presence of E. coli EM (A) or S. epidermidis EM (B) or with E. coli LPS as a positive control, as indicated. Cells were then stained with fluorescent antibodies, washed, and analyzed by flow cytofluorimetry. Data are the means ± SEs (n = 3, run in triplicate). Black asterisks, P < 0.05 with respect to cells treated with NPs alone for all EM concentrations used; green, red, or blue asterisks, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green, red, and blue symbols, respectively). Orange straight lines, IC50 values; red boxes, the induction of marker expression occurred at concentrations less than the IC50 values.
- FIG 5
Mixed lymphocyte reaction (MLR) between allogeneic T lymphocytes and DCs pretreated with SiO2 NPs and bacterial EMs. Allogeneic T cells, prelabeled with CFSE, were incubated for 5 days with DCs that had been preincubated with the indicated doses of SiO2 NPs and EMs for 24 h and washed before the assay. The percentages of proliferating T cells, identified on the basis of their reduced MFI by FACS, are the means ± SEs (n = 7, run in triplicate); the concentration of LPS, which was used as a positive control, was 100 ng/ml. Black asterisks, P < 0.05 with respect to cells treated with NPs alone for all EM concentrations used; green or red asterisks, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green and red symbols, respectively). Orange straight lines, IC50 values.
- FIG 6
Synergic induction of a DC population unable to overexpress CD86 by SiO2 NPs and bacterial EMs. (A) Illustrative dot plots and histogram plots of CD86 expression distributions obtained by FACS analysis of DCs treated for 24 h with SiO2 NPs and EMs. Gating, the percentage of anomalous cells (green) with reduced side and forward scattering properties, and their corresponding levels of CD86 expression (indicated as MFI values) are shown. Red histogram plots represent functional DCs with forward scatter (FSC) and side scatter (SSC) parameters similar to those of control (Ctrl) cells; green histogram plots represent anomalous dendritic cells that had forward scatter and side scatter parameter values lower than those of control cells and that were unable to undergo maturation. Data are from 1 representative experiment of 10 experiments conducted. (B) Illustrative histogram plots of the CD86 MFI expressed by dendritic cells after 24 h of incubation with the different stimuli indicated in the figure. Numbers indicate the MFI of the peak. Data represent the percentage of altered DCs (with low forward scatter and side scatter parameter values) after treatment with different NP and EM concentrations. (C) CD86 expression (indicated as MFI values) by total cells (with no gating in dot plots), normal cells (gated by means of their forward scatter and side scatter values similar to those for control cells), and shrunken cells (gated by means of their low forward scatter and side scatter values) after DC treatment (with NPs alone or with different concentrations of EMs). S. epi, S. epidermidis.
- FIG 7
Percentages of CD4+ and CD8+ cells and cytokine release by allogeneic T lymphocytes mixed with DCs pretreated with SiO2 NPs and bacterial EMs. (A) The relative amounts of CD4+ T cells and CD8+ T cells among resting or DC-induced proliferating lymphocytes after preincubation with LPS or NPs and EMs were determined by FACS analysis using specific antibodies (means ± SEs; n = 4, run in triplicate). *, P < 0.05 with respect to control cells. (B) Relative amounts of cytokines produced by allogeneic T lymphocytes after 5 days of incubation with DCs preincubated for 24 h with SiO2 NPs and bacterial EMs, as indicated. Data are from one representative experiment of four experiments conducted and represent means ± SEs. Black asterisks, P < 0.05 with respect to cells treated with NPs alone for all the EM concentrations used; green or red asterisks, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green and red symbols, respectively); green, blue, or red section signs, P < 0.05 with respect to cells treated with NPs alone for the indicated concentration of EMs (indicated by the green, blue, and red symbols, respectively). Orange straight lines, IC50 values; red boxes, induction of cytokine release occurred at concentrations less than the IC50 values.
- FIG 8
SiO2 NP uptake by DCs is not affected by the presence of bacterial EMs. (A) After incubation at 37°C with FITC-labeled Stöber SiO2 NPs, bacterial EMs, or E. coli LPS as a positive control, NP uptake by DCs was quantified by flow cytofluorimetry. Data, expressed as MFI, are means ± SEs (n = 2, run in duplicate). The percentage of positive cells is indicated in brackets. *, P < 0.05 with respect to samples treated with NPs alone. (B) DCs were placed on poly-l-lysine-coated slides and left untreated in the heated chamber for 5 min to allow adhesion to the surface of the slides; then, the cells were incubated with FITC-labeled Stöber NPs and bacterial EMs, as indicated, for 2 h, washed, and directly analyzed by confocal microscopy.
- FIG 9
Dependence of NP and EM synergy on proteinaceous PAMPs and on an association with bacterial factors on the surface of NPs. (A) DCs were treated (for 24 h at 37°C) with EMs or with EMs that had been incubated for 1 h at 50°C with 100 μg/ml proteinase K and then for 5 min at 95°C in order to degrade proteins; the cells were then analyzed for CD86 expression (top), and the amount of IL-1β in the culture supernatant was measured (bottom); data are from one representative experiment of three experiments conducted and represent means ± SEs. *, P < 0.05 with respect to cells not treated with proteinase K. (B) NPs (100 μg/ml) were incubated with the indicated EM concentrations for 20 min at 37°C in RPMI 1640 containing 10% FBS. The NPs were then centrifuged for 30 min at 13,000 rpm, and the supernatant was recovered. The NPs were washed three times with PBS, and finally, some NPs were resuspended in fresh medium and other NPs were resuspended with the recovered supernatant. Then, the NPs were used to treat DCs for 24 h at 37°C and IL-1β production was measured by ELISA. Data are from one representative experiment of three experiments conducted and represent means ± SEs. *, P < 0.05. I, NPs coincubated with the indicated medium and DCs; II, NPs preincubated with the indicated medium, pelleted, and incubated with DCs after resuspension in new medium without EM; III, NPs preincubated with the indicated medium, pelleted, and incubated with DCs after resuspension in their corresponding supernatants.
Tables
- TABLE 1
Summary of synergistic activity of EMs and SiO2 NPs on DCsa
↵a The increase in the levels of cytokine production and maturation marker induction by DCs and the T lymphocyte-activating capacity (proliferation, phenotype differentiation [diff], and cytokine secretion) due to EM and NP costimulation were correlated with cytotoxic phenomena. Changes occurring at cytotoxic NP doses are highlighted in red. The following semiquantitative notations are used to help the reader recall the rough intensity of the parameters analyzed: +/−−, very weak effect; +/−, weak effect; +, an effect; ++, a strong effect; ND, not determined.
Supplemental material
- Supplemental file 1 -
Fig. S1. TEM micrographs and size distribution of Ludox TM-40 amorphous silica nanoparticles and FITC-labeled Stöber SiO2-NPs. Table S1. Protein concentrations of undiluted EM from indicated bacterial cultures and calculated after the dilutions used in the study. Fig. S2. The extracellular media (EM) obtained from overnight culture of the indicated bacteria or Luria-Bertani culture medium, as control, was serially diluted as indicated in RPMI medium plus 10% FBS and incubated with DCs for 24 h. Fig. S3. DCs were incubated for 3 h with SiO2-NPs alone or in the presence of EM or LPS as positive control. Fig. S4. Peripheral blood monocytes were incubated for 24 h with SiO2-NPs and bacterial EM as indicated. Fig. S5. (A) DCs, incubated for 24 h with SiO2-NPs, EM, and LPS (positive control) as indicated, were collected, stained with anti-CD83 and -CD86 antibodies, washed, and analyzed by flow cytofluorimetry. (B) Background signal at different fluorescent excitation channels of dendritic cells treated with different concentrations of Ludox NPs. Fig. S6. MLR assay indicating T cell percent proliferation after 5 days, mediated by DCs pretreated for 24 h with no agonists or LPS at the indicated doses, or due to PHA (phytohemagglutinin; positive control). Fig. S7. Illustrative dot plots of dendritic cells after 24-h incubation with different stimuli. Fig. S8. CD86 expression distributions obtained by FACS analysis of DCs treated for 24 h with SiO2-NPs and EMs. Fig. S9. Cytokine patterns after MLR. Fig. S10. DCs were incubated with FITC-labeled Stöber SiO2-NPs at 37°C or at 0°C (on melting ice) and at doses and times indicated.
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- Supplemental file 1 -
