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

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

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

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

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

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

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk

<div><p>Background</p><p>Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.</p><p>Objective</p><p>To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV.</p><p>Methods</p><p>ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. <i>S. Epidermidis</i> and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.</p><p>Results</p><p>bIgG recognised human RSV, influenza haemagglutinin and <i>Haemophilus influenza</i>. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.</p><p>Conclusions</p><p>The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.</p></div

TNFα and IL-10 production by THP-1 macrophages stimulated with 2 mg/ml intact whey protein or whey hydrolysate in the presence or absence of celastrol.

<p>Celastrol significantly inhibited both TNFα and IL-10 induced by intact whey protein and whey hydrolysates, demonstrating NF-κB dependency of the cytokine production. Significant differences were determined by using paired T tests. Significant differences were indicated by *.</p

NF-kB/AP-1 expression in THP-1-MD2-CD14 and THP-1-MyD88 deficient reporter cells after stimulation with intact whey and casein proteins or hydrolysates.

<p>Intact whey and the two most mildly hydrolyzed whey hydrolysates induced TLR signaling, while casein samples had no effect (left panel). These effects were TLR dependent, since no activation was observed in the MyD88 deficient cell line after stimulation (right panel). Significant differences compared to the negative control were determined by using the Kruskal-Wallis test followed by the Dunn’s test and indicated by *.</p

Bovine Ig enhances internalisation of hRSV by hPMN.

<p>GFP-renilla expressing RSV was pre-incubated with medium in the presence or absence of IVIg or bIgG and allowed to bind to PMN at 4°C. Subsequently, cells were incubated at 37°C and thereafter treated with trypsin and acid to remove extracellular RSV. Cells were then washed and analysed by flow cytometry for the percentage of GFP+ cells. GFP+ cells were tested in the absence of RSV (–), in the presence of RSV but absence of Ig (0) and in the presence of IVIg or bIgG (µg/ml). Mean and S.E.M. of triplicate measurements of one out of five donors tested are shown.</p