LAB-primed MoDC stimulated with scGOS/lcFOS are characterized by an enhanced release of IL-10 while no enhanced IL-12p70 secretion is detectable.

<p>Immature human MoDC were incubated with <i>B</i>.<i>breve</i> (clear dots, A, B) or <i>L</i>.<i>rhamnosus</i> (clear triangle, C, D) in different concentrations with (black marks) or without (clear marks) scGOS/lcFOS (5 mg/ml) for 24h. Supernatants were analyzed for secretion of IL-10 (A, C) and IL-12p70 (B, D) by ELISA. Results are presented as mean ± SEM, ten independent experiments are shown. * p<0.05, ** p<0.01, Mann Whitney test. LAB = Lactic acid bacteria, MoDC = monocyte-derived dendritic cells.</p

Stimulation with scGOS/lcFOS directly induces a dose dependent IL-10 release by MoDC that is impaired by addition of a TLR4 antagonist.

<p>Immature human MoDC were exposed to oligosaccharide mixture scGOS/lcFOS (312 μg/ml–5 mg/ml) for 24h in the presence (black marks) or absence (grey marks) of a TLR4 antagonist. The amount of IL-10 was measured by ELISA in cell-free supernatant. Results are presented as mean ± SEM, five independent experiments are shown. * p<0.05, Mann Whitney test. scGOS/lcFOS = short chain galacto-, long chain fructo-oligosaccharide-mixture, MoDC = monocyte-derived dendritic cells.</p

scGOS/lcFOS stimulated MoDC induce CD4⁺Foxp3⁺ T cells.

<p>MoDC stimulated with scGOS/lcFOS (5 mg/ml) were used to prime naïve CD4⁺CD45RA⁺ T cells in an ASA. At day 7, cells were stained for CD4 and intracellular Foxp3 was analyzed by flow cytometry. The dot plots show data from one representative experiment (A-C). T cells that have been co-incubated with medium-treated MoDC served as medium/negative control (A) and a positive control for Treg polarization is shown in (B). CD4⁺Foxp3⁺ T cells induced by scGOS/lcFOS stimulated MoDC are shown in (C). Data in (D) are expressed as percentages of CD4⁺Foxp3⁺ cells. Results are presented as mean ± SEM, 4 independent experiments are shown (D). * p<0.05, Mann Whitney test. ASA = allogeneic stimulation assay, scGOS/lcFOS = short chain galacto-, long chain fructo-oligosaccharides, MoDC = monocyte-derived dendritic cells.</p

MoDC primed with scGOS/lcFOS are characterized by release of IL-10 while no IL-12p70 secretion is detectable.

<p>Immature human MoDC were incubated with scGOS/lcFOS (5 mg/ml) for 24h. Medium-treated MoDC served as negative control, MoDC matured by LPS as positive control for functional MoDC stimulation. Supernatants were analyzed for secretion of IL-10 (A) and IL-12p70 (B) by ELISA. Results are presented as mean ± SEM, ten independent experiments are shown. **** p<0.0001, ** p<0.01, unpaired student’s t test. scGOS/lcFOS = short chain galacto-, long chain fructo-oligosaccharides, MoDC = monocyte-derived dendritic cells.</p

scGOS/lcFOS-stimulated MoDC release cytokines and chemokines in a different manner compared to LPS.

<p>MoDC were stimulated with scGOS/lcFOS (5 mg/ml) for 24h. MoDC matured by LPS served as positive control for functional stimulation. The amount of cytokines and chemokines in cell-free supernatants was measured by luminex-based assay (readout: IL-1α, IL-1β, IL-6, IL-23p19, IL-27, TNFα, IP-10, MDC (CCL22), TARC (CCL17), MIP3α (CCL20) and ELISA (IL-10, IL-12p70)). Results are presented in pg/ml. Three independent experiments are shown.</p

Suppressive capacity of T cells induced by scGOS/lcFOS-treated MoDC.

<p>T cells of allogeneic stimulation assays (ASA) were harvested on day seven and co-incubated with freshly isolated, violet-labeled responder CD4⁺ T cells a ratio of 1:1 for five days in presence of anti-CD3/28 and rhIL-2. The mix of ASA cells and violet stained responder CD4⁺ T cells was then stained (after five days of co-incubation) with CD4 APC-Cy7. The dot plots show data from one representative experiment. Proliferation of responder T cells only (A) or in co-incubation with T cells induced by medium- (B), LPS- (C) and scGOS/lcFOS- (E) stimulated MoDC and Treg control (D) is shown. Results in (F) are presented as mean ± SEM, 4 independent experiments are depicted. Proliferation of violet-positive cells was analyzed by flow cytometry and suppressive functionality index was determined by calculating dividing cells (div)/ non-dividing (ndiv) cells (F). * p<0.05, Mann Whitney test. ASA = allogeneic stimulation assay, scGOS/lcFOS = short chain galacto-, long chain fructo-oligosaccharides, MoDC = monocyte-derived dendritic cells, div = dividing cells, ndiv = non-dividing cells.</p

Comparison of fungal pattern on pollen from birch and timothy grass.

<p><b>A)</b> PCoA (Bray-Curtis-Index) of fungal pattern on birch pollen (<i>Betula pendula</i>; n = 31; black) and timothy grass pollen (<i>Phleum pratense</i>; n = 16; grey) show hardly overlapping 95%-confidence. The significant differences were confirmed by ANOSIM (R = 0.51, p = 0.0001). <b>B)</b> The cluster dendrogramm (Bray-Curtis-Index) demonstrates the separation of the two pollen species due to fungal pattern. <b>C,D)</b> Boxplots of indices weighing species due to their extent of abundance in fungal community on birch- and timothy grass-pollen. In comparison the two groups differed significantly due to low abundant species (Shannon-index, p = 0.006), and also due to high abundant species of fungal composition (Simpson-index, p = 0.005).</p

Comparison of bacterial and fungal pattern on pollen collected in rural and urban sample sites.

<p>PCoA performed to compare the microbial composition on pollen obtained from rural (grey) and urban areas (black). <b>A)</b> Bacterial composition on birch pollen. <b>B)</b> Bacterial composition on timothy grass pollen. <b>C)</b> Fungal composition on birch pollen. <b>D)</b> Fungal composition on timothy grass pollen.</p

Spearman correlations of bacterial diversity on birch pollen to allergenicity parameter.

<p>Scatterplot including linear trend line, spearman correlation values rho and significance level p of diversity indices (Shannon H/Simpson 1-D) and parameter (number of fragments n(tRFs)) of bacterial pattern on birch pollen (<i>Betula pendula</i> 2014, n = 55) with allergen concentration in pollen. <b>A-D)</b> Correlation of n(tRFs) and allergenicity parameters Bet v 1 (r_s = 0.28, p = 0.038), PALM_LTB4 (r_s = 0.14, p = 0.33), PALM_PGE2 (r_s = 0.17, p = 0.22) and NADPH oxidase (r_s = -0.04, p = 0.79). <b>E-H)</b> Correlation of Simpson diversity Index 1-D and allergenicity parameters Bet v 1 (r_s = 0.25, p = 0.068), PALM_LTB4 (r_s = 0.08, p = 0.53), PALM_PGE2 (r_s = .12, p = 0.366) and NADPH oxidase (r_s = 0.2, p = 0.19). <b>I-L)</b> Correlation of Shannon diversity Index H and allergenicity parameters Bet v 1 (r_s = 0.14, p = 0.317), PALM_LTB4 (r_s = -0.36, p = 0.006), PALM_PGE2 (r_s = -0.04, p = 0.0015) and NADPH oxidase (r_s = 0.2, p = 0.19).</p

Comparison of bacterial pattern on pollen from birch and timothy grass.

<p><b>A)</b> PCoA (Bray-Curtis-Index) of bacterial pattern on birch pollen (<i>Betula pendula</i>; n = 55; black) and timothy grass pollen (<i>Phleum pratense</i>; n = 20; grey) show hardly overlapping 95%-confidence intervals. The significant differences were confirmed by ANOSIM (R = 0.81, p = 0.0001). <b>B)</b> In the cluster dendrogramm (Bray-Curtis) pollen from both species are forming separated groups also attesting the significant difference. <b>C,D)</b> Boxplots of indices weighing species due to their extent of abundance in bacterial community on birch- (<i>Betula pendula</i>; n = 55) and timothy grass-pollen (<i>Phleum pratense</i>, n = 20). In comparison the two groups differs significantly due to low abundant species (Shannon-index, p = 0.009), but not significantly due to high abundant species of bacterial composition (Simpson-index, p = 0.78).</p