The oligosaccharides 6’-sialyllactose, 2’-fucosyllactose or galactooligosaccharides do not directly modulate human dendritic cell differentiation or maturation

<div><p>Breast milk plays an important role in immune development in early life and protects against diseases later in life. A wide range of the beneficial effects of breast milk are attributed to human milk oligosaccharides (HMOs) as well as components such as vitamin D3 (VitD3) or TGFβ. One mechanism by which HMOs might contribute to immune homeostasis and protection against disease is the induction of a local tolerogenic milieu. In this study we investigated the effect of the HMOs 6’-sialyllactose (6’SL) and 2’-fucosyllactose (2’FL) as well as prebiotic galactooligosaccharides (GOS) on DC differentiation and maturation. Isolated CD14+ monocytes were cultured for six days in the presence of GM-CSF and IL-4 with or without 6’SL, 2’FL, GOS, VitD3 or TGFβ. Additionally, immature VitD3DC, TGFβDC and moDC were used as different DC types to investigate the effect of 6’SL, 2’FL and GOS on DC maturation. Surface marker expression and cytokine production was measured by flow cytometry and cytometric bead array, respectively. Unlike TGFβ and vitD3, the oligosaccharides 6’SL, 2’FL and GOS did not influence DC differentiation. Next, we studied the effect of 6’SL, 2’FL and GOS on maturation of moDC, VitD3DC and TGFβDC that showed different profiles of HMO-binding receptors. 6’SL, 2’FL and GOS did not modulate LPS-induced maturation, even though their putative receptors were present on the different DCs types. Thus, whereas VitD3 and TGFβ halt DC differentiation, which results in phenotypically distinct tolerogenic DCs, 6’SL, 2’FL and GOS do not alter DC differentiation or maturation of <i>in vitro</i> differentiated DC types.</p></div

TGFβ and VitD3 differentially modulate the production of IL-6 and IL-8 during differentiation.

<p>6’SL was treated with an optimized Triton X-114 method to remove LPS traces. Immature DC were cultured for six days in the presence or absence of VitD3, TGFβ, 6’SL, LPS-free 6’SL, 2’FL or GOS. A,C) IL-6 and B,C) IL-8 were measured in the supernatant by CBA.</p

moDC, TGFβDC and VitD3DC express different levels of HMO-recognizing receptors.

<p>The expression of several receptors that are shown in literature to recognize HMOs were measured on A) monocytes and B-G) moDC, TGFβDC and vitD3DC. The expression of Siglec-5, Siglec-7, TLR4, CD206, Galactin-3 and DC-SIGN (open histograms) and their matching isotype control (shaded histograms) were measured on monocytes. The expression of B) CD206, C) DC-SIGN, D) Siglec-5, E) TLR4, F) Galactin-3 and G) Siglec-7 was shown by scatter plots (n = 3–5) and histogram of one representative donor. Normal distribution was assumed due to the low sample size. Significance was tested by a repeated measures ANOVA with a Tukey’s multiple comparison post-hoc test.</p

TGFβDC and VitD3DC induce tolerogenic DC.

<p>Immature DC were stimulated with LPS for 48 hours. A) The expression of CD83, PD-L1, CD80 and CD86 on immature DC (shaded histograms) or mature DC (open histograms) of one donor was shown. The relative surface marker expression of B) CD83, C) CD86 and E) CD80 are shown. Relative fold change was calculated by dividing the MFI (median fluorescence intensity) of DC differentiated in the presence of a breast milk component/MFI of moDC of each respective donor. D) Percentage of CD86+CD83+ DCs. F) IL-12p70 and IL-10 and TNF were measured in the supernatant by CBA. G) The IL-10/TNF ratio is shown for the different mature DC that were differentiated in the presence of different breast milk components.</p

HMOs do not impact maturation of different in vitro generated DC.

<p>Immature moDC, TGFβDC and VitD3DC were stimulated with LPS with or without 6’SL, 2’FL or GOS for 48 hours. The effect of 6’SL, 2’FL and GOS was measured in the absence (A, C, F) or presence of LPS (B, D,F). The surface marker expression of A, B) PD-L1 and C, D) CD80 and E, F) CD83 is shown as MFI (median fluorescence intensity) (two independent experiments, six donors).</p

TGFβ and VitD3 induce phenotypic distinct DCs.

<p>CD14+ monocytes were cultured in the presence of IL-4 and GM-CSF for six days in the presence or absence of breast milk components. Surface marker expression was measured by flow cytometry. A) A multi-colour overlay of CD14 expression versus CD1a expression on moDC (black), TGFβDC (blue) or VitD3 (red) of one representative donor is shown. The percentage of B) CD14+ and C) CD1a+ DC and relative surface marker expression of D) HLA-DR, E) CD80, F) CD86 or G) PD-L1 on immature DC differentiated in the presence of TGFβ, VitD3, 6’SL, 2’FL or GOS was shown. Relative fold change was calculated by dividing the MFI (median fluorescence intensity) of DC differentiated in the presence of a breast milk component/MFI of moDC of each respective donor.</p

Extraction of remaining TX-114 from protein extract with high-affinity Bio-Beads results in most effective lowering of detergent concentration to non-toxic levels.

<p>Comparison of different TX-114 extraction methods. Starting from an initial TX-114 concentration of ~ 2% (v/v), the concentration of this detergent is effectively lowered by the application of dialysis, various centrifugation conditions, spin-X column and Bio-Bead assisted purification. Application of Bio-Beads results in most efficient TX-114 extraction down to 0.005% (v/v).</p

Optimization of TX-114 removal method from beta-lactoglobulin (BLG) and soy protein extract (SPE).

<p><i>(A)</i> Triton concentrations can be quantified in protein-free solutions using spectroscopic absorbance at 280 nm. Absorbance spectrum of 0.005% (v/v) TX-114 solution in PBS was determined by a NanoDrop ND1000 spectrophotometer. <i>(B)</i> TX-114 in PBS dose-dependent absorption at 280 nm. Results are corrected for PBS background and represent an average of three independent measurements. <i>(C)</i> Concentration of TX-114 in PBS spiked with LPS (0.45 EU/l) after applying the TX-114 treatment described in Materials and Methods measured after one TX-114 cycle, three TX-114 cycles or after one TX-114 cycle followed with Bio-Beads treatment. <i>(D)</i> TX-114 reduces LPS detection with EndoZyme recombinant factor C assay in a dose-dependent manner. LPS concentration was measured in PBS spiked with 0.45 EU/l of LPS and decreasing concentration of TX-114. <i>(E)</i> Concentration of TX-114 in the medium equal or higher than 0.006% (v/v) decreases the viability of THP-1 derived macrophages. Viability of THP-1 macrophages cultured for 24 h in the presence of TX-114 in the medium expressed as relative to cells grown in TX-114 free medium = 1).</p

THP-1 derived macrophages as a model to study immunomodulatory potential of non-purified and LPS-purified BLG preparation.

<p><i>(A)</i> LPS dose-dependence and differential gene expression of inflammatory cytokines and NF-κB in THP-1 macrophages. The data obtained for 3 repetitions is normalized to GAPDH and relative to unstimulated macrophages cultured in medium. Statistically significant differences relative to non-stimulated THP-1 macrophages were calculated with Student’s <i>t</i>-tests: * = P<0.05. <i>(B)</i> LPS dose-dependent secretion of IL-8 by THP-1 macrophages. THP-1 cells were incubated 24h with increasing concentration of LPS and IL-8 concentration in supernatant was determined with flow cytometry. The results and statistically significant differences are related to unstimulated cells medium control = 1). <i>(C-E)</i> Dose dependent secretion of pro-inflammatory cytokines from THP-1 macrophages incubated 24h with non-purified BLG (BLG) and BLG purified with TX-114 method (TX BLG). The results are expressed as the relative to unstimulated cells (medium control = 1). Statistically significant differences between corresponding dilutions of purified and non-purified BLG preparations are shown (p<0.05).</p

Correlation of staining patterns of all bands stained by all isotypes (A) or stained by IgTotal (B), IgM (C), and IgG (D) autoantibodies to CLL.

<p>Chickens were one year of age and divergently selected for 29 generations for High (red circles: Hg), Low (green diamonds: Lg), and Control (purple squares: Cg) agglutination titres to SRBC at 5 weeks of age, and C line birds phenotypically selected for high (yellow bars: Ch) or low (blue bars: Cl) NAb levels to KLH at 16 weeks of age. Ordination plots by PCA. Graphs include 11–12 birds from each line as also used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072276#pone-0072276-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072276#pone-0072276-g002" target="_blank">2</a>. Only CLL fragments that showed significant line differences for staining intensity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072276#pone-0072276-t004" target="_blank">Tables 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072276#pone-0072276-t006" target="_blank">6</a>) are shown. ‘Eigenwaardes’ were 12.8% horizontal and 11.1% vertical axes (A), 17.9% horizontal and 11.6% vertical axes (B),16.5% horizontal and 15.4% vertical axis (C), and 20.8% horizontal and 12.5% verical axis (D), respectively. Numbers in <i>Ithalic</i> represent CLL fragments most informative for line clustering.</p