Maturation of DCs induced by particle-bound PADRE is via sphingosine kinase but not PLC.

<p>Effect of the PLC inhibitor D609 (<b>A</b>) and the sphingosine kinase inhibitor SKI-II (<b>B</b>) on the phenotypic maturation of DCs induced by particle-bound PADRE. Day-5 DCs were incubated with the inhibitors for 2 hours and then cultured with particle-bound PADRE, particle-bound P2C or ionomycin for 2 days and analyzed by flow cytometry for CD80, CD86, CD83 and HLA-DR. The histograms show the expression levels of a representative donor of at least three independent experiments done with cells from three different healthy donors.</p

Direct Activation of Human Dendritic Cells by Particle-Bound but Not Soluble MHC Class II Ligand

<div><p>Dendritic cells (DCs) are key activators of cellular immune responses through their capacity to induce naïve T cells and sustained effector T cell responses. This capacity is a function of their superior efficiency of antigen presentation via MHC class I and class II molecules, and the expression of co-stimulatory cell surface molecules and cytokines. Maturation of DCs is induced by microbial factors via pattern recognition receptors such as Toll-like receptors, pro-inflammatory cytokines or cognate interaction with CD4⁺ T cells. Here we show that, unexpectedly, the PanDR helper T cell epitope PADRE, a generic T helper cell antigen presented by a large fraction of HLA-DR alleles, when delivered in particle-bound form induced maturation of human DCs. The DCs that received the particle-bound PADRE displayed all features of fully mature DCs, such as high expression of the co-stimulatory molecules CD80, CD86, CD83, the MHC-II molecule HLA-DR, secretion of high levels of the biologically active IL-12 (IL-12p70) and induction of vigorous proliferation of naïve CD4⁺ T cells. Furthermore, the maturation of DCs induced by particle-bound PADRE was shown to involve sphingosine kinase, calcium signaling from internal sources and downstream signaling through the MAP kinase and the p72syk pathways, and finally activation of the transcription factor NF-κB. Based on our findings, we propose that particle-bound PADRE may be used as a DC activator in DC-based vaccines.</p></div

Maturation of DCs induced by particle-bound PADRE involves CaMKII.

<p>Effect of the CaMKII inhibitor CK59 on the phenotypic maturation of DCs induced by particle-bound PADRE. Day-5 DCs were cultured with particle-bound PADRE, particle-bound P2C or ionomycin for 2 days and analyzed by flow cytometry for CD80, CD86, CD83 and HLA-DR. The histograms show the expression levels of a representative donor of four independent experiments done with cells from four different healthy donors.</p

DCs stimulated with particle-bound PADRE induce vigorous proliferation of naïve CD4⁺ T cells.

<p>Adherent PBMCs previously depleted of T cells were cultured in presence of GM-CSF and IL-4 for five days and matured by incubation with particles carrying PADRE, P2C, a mixture of both, or a mixture of particles carrying either PADRE or P2C. After two days, the DCs were washed characterized by flow cytometry as for the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063039#pone-0063039-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063039#pone-0063039-g002" target="_blank">2</a> and then co-cultured with CFSE-labelled naïve CD4⁺ T cells. After seven days of co-culture, the cells were harvested and labelled with antibodies to CD4, CD45RA and CD45RO and analyzed by flow cytometry. Proliferation of the T cells was determined by the reduction of CFSE upon successive cell divisions. The plots show CFSE and CD45RA (<b>A</b>) or CD45RO (<b>B</b>) on gated CD4⁺ T cells of one of five independent experiments with cells from five different healthy donors.</p

Specificity of the DC-inducing activity of particle-bound PADRE.

<p>iDCs as in the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063039#pone-0063039-g001" target="_blank">Figure 1</a> were incubated either without stimuli (negative control) or with an inflammatory cytokine cocktail of IL-1β, IL-6 and TNF-α as positive controls, or with particles without cargo or loaded with GILGFVFTL peptide, NLVPMVATV peptide, PADRE, diacyl-lipopeptide P2C, a mix of both PADRE and P2C or with a mixture of particles carrying either PADRE or P2C. After two days cultures the cells were labelled with antibodies for the CD11c and Lin (cocktail of FITC-conjugated antibodies against the molecules CD3, CD14, CD19 and CD56) for exclusion, and CD80, CD83, CD86 and HLA-DR, and analyzed by flow cytometry. The graphs show fold of expression of the molecules CD80 (<b>A</b>), CD86 (<b>B</b>), CD83 (<b>C</b>) and HLA-DR (<b>D</b>) with standard error. n = 6. * = p<0.05; ** = p<0.01; and *** = p<0.001 comparing the group treated with the indicated compound vs. the group “No Treatment”.</p

Particle-bound PADRE triggers DC maturation by inducing release of calcium from intracellular sources.

<p>Mobilization of intracellular calcium in DCs induced by particles carrying PADRE. iDCs generated as in the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063039#pone-0063039-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063039#pone-0063039-g002" target="_blank">2</a> were harvested on day 5 and labelled with Fluo-3/AM. The DCs were then suspended in medium with or without EDTA and incubated for 10 minutes with empty particles, particle-bound PADRE, particle-bound P2C, soluble LPS or ionomycin and analyzed by flow cytometry. The graph shows mean fold changes of Fluo-3 fluorescence with standard error of independent experiments done with cells from three to five different healthy donors (<b>A</b>)<b>.</b> *indicates p<0.05– none vs. stimulus. In (<b>B–C</b>) is shown the effect of the blockade of intracellular calcium on the phenotypic maturation and IL-12p70 secretion of DCs induced by particle-bound PADRE. 5-day DCs were harvested, labelled or not with BAPTA-AM and incubated with particle-bound PADRE, particle-bound P2C or ionomycin for 2 more days. On day 7 the cells were labelled with antibodies for the CD80, CD86, CD83 and HLA-DR, and analyzed by flow cytometry. A representative experiment is shown (<b>B</b>). The supernatant of the stimulation cultures were collected and analyzed for IL-12p70 by ELISA (<b>C</b>). n = 3.</p

Effect of the specific inhibitors of kinase signaling on the phenotypic maturation of DCs induced by particle-bound PADRE.

<p>Day-5 DCs were incubated for 2 hours with inhibitors for p72 Syk, PI3K, p38 MAPK or NF-κB after which particle-bound PADRE, particle-bound P2C or soluble LPS were added and the cultures continued for 2 more days. On day 7 the cells were analyzed by flow cytometry for expression of CD80, CD86, CD83 and HLA-DR and their culture supernatant by ELISA for IL-12p70. The graphs show fold change of expression of CD80 (<b>A</b>), CD86 (<b>B</b>), CD83 (<b>C</b>) and HLA-DR (<b>D</b>), or IL-12p70 levels in the supernatants (<b>E</b>) with standard error of 7 independent experiments with cells from 7 different healthy donors. * = p<0.05; ** = p<0.01; and *** = p<0.001 comparing the group treated with stimulus only vs. the groups treated with stimulus plus the specific inhibitor.</p

Phenotypic maturation of dendritic cells (DCs) induced by particles coated with PADRE.

<p>iDCs were generated from adherent PBMCs depleted of T cells. The adherent cells were cultured in serum-free AIM-V medium with GM-CSF and IL-4, harvested on day 5 and incubated with soluble PADRE, particle-bound PADRE or an inflammatory cytokine cocktail of IL-1β, IL-6 and TNF-α for 2 additional days. On day 7 the cells were labelled with antibodies for the HLA-DR, CD11c and Lin (cocktail of FITC-conjugated antibodies against the molecules CD3, CD14, CD19 and CD56) for exclusion, and CD80, CD83 and CD86, and analyzed by flow cytometry. The graphs show mean fold changes with standard error of the expression of CD80 (<b>A</b>), CD86 (<b>B</b>), CD83 (<b>C</b>), and HLA-DR (<b>D</b>). n = 5; NT: no treatment; Cocktail: IL-1β, IL-6 and TNF-α. * = p<0.05; ** = p<0.01; and *** = p<0.001 comparing the group treated with the indicated compound vs. the group “NT”.</p

Maturation of DCs induced by particle-bound PADRE does not involve calcineurin.

<p>Day-5 DCs were collected and incubated with particle-bound PADRE, particle-bound P2C or ionomycin for additional 2 days in the presence of absence of cyclosporine A. On day 7 the cells were stained with antibodies for CD80, CD86, CD83 and HLA-DR, and analyzed by flow cytometry. The culture supernatants were analyzed for IL-12p70 by ELISA. The histograms in (<b>A</b>) show the expression of CD80, CD86, CD83 and HLA-DR of a representative donor, diagram (<b>B</b>) shows the secretion of IL-12p70. Independent experiments were done with cells from seven different healthy donors.</p

Antiseptic treatment of <i>Pseudomonas aeruginosa</i> SG81 biofilms.

<p>The analytical results by the Number of samples (n), Colony reduction factor (CRF) in log₁₀ (CFU/cm²) ± Standard Deviation (SD), lower and upper 95% confidence limits (CI) after exposure to air plasma for 30–600 s treatment time respectively and 0.1% CHX after 600 s exposure time and untreated control of <i>Pseudomonas aeruginosa</i> SG81 biofilms [p-values of omnibus tests (Kruskal-Wallis) and two-sample tests (Whitney <i>U</i>); statistical significance: α = 0.05].</p>c<p>significantly different from CHX.</p>*<p>significantly different from the respective treatment time of <i>Staphylococcus epidermidis</i> RP62A.</p