Dietary Vitamin D Supplementation Is Ineffective in Preventing Murine Cow's Milk Allergy, Irrespective of the Presence of Nondigestible Oligosaccharides.

INTRODUCTION: Cow's milk allergy (CMA) is one of the most common food allergies especially early in life. A mixture of nondigestible short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides, and pectin-derived acidic-oligosaccharides (GFA) may reduce allergy development and allergic symptoms in murine CMA. Recently, vitamin D (VitD) has been suggested to have beneficial effects in reducing allergy as well. OBJECTIVE: In this study, the immune modulatory effect on allergy prevention using the combination of GFA and VitD was investigated. METHODS: Female C3H/HeOuJ mice were fed a control or GFA-containing diet with depleted, standard (1,000 IU/kg), or supplemented (5,000 IU/kg) VitD content for 2 weeks before and during whey sensitization (n = 10-15). Mice were sensitized 5 times intragastrically with PBS as a control, whey as cow's milk allergen, and/or cholera toxin as adjuvant on a weekly interval. One week after the last sensitization, mice were intradermally challenged in both ear pinnae and orally with whey, subsequently the acute allergic skin response and shock symptoms were measured. After 18 h, terminal blood samples, mesenteric lymph nodes, and spleens were collected. Whey-specific immunoglobulin (Ig) E and IgG1 levels were measured by means of ELISA. T cell subsets and dendritic cells (DCs) were studied using flow cytometry. RESULTS: Additional VitD supplementation did not lower the allergic symptoms compared to the standard VitD diet. CMA mice fed the GFA diet supplemented with VitD (GFA VitD+) significantly decreased the acute allergic skin response of whey sensitized mice when compared to the CMA mice fed VitD (VitD+) group (p < 0.05). The effect of GFA was not improved by extra VitD supplementation even though the CMA mice fed the GFA VitD+ diet had a significantly increased percentage of CD103+ DCs compared to the VitD+ group (p < 0.05). The VitD-deprived mice showed a high percentage of severe shock and many reached the humane endpoint; therefore, these groups were not further analyzed. CONCLUSIONS: High-dose VitD supplementation in mice does not protect against CMA development in the presence or absence of GFA.

IL-10 Receptor or TGF-ß Neutralization Abrogates the Protective Effect of a Specific Nondigestible Oligosaccharide Mixture in Cow-Milk-Allergic Mice.

Background: Dietary nondigestible, short-chain galacto-, long-chain fructo-, and pectin-derived acidic oligosaccharides (GFAs) lower the effector response in cow-milk-allergic (CMA) mice; and forkhead box P3 (Foxp3)-positive regulatory T cells (Tregs) were shown to contribute to this. Objective: The aim of this study was to assess the contribution of interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß) to the protective effect of the GFA diet in CMA mice. Methods: Female C3H/HeOuJ mice, 3-4 wk old, were orally sensitized with cholera toxin (Sham) or whey and cholera toxin (Whey) 1 time/wk for 5 consecutive weeks and challenged with whey 1 wk later. The mice were fed a control or 1% GFA (9:2:1) (Whey+GFA) diet starting 2 wk before the first sensitization. In a second experiment, the mice were also injected with αIL-10 receptor (αIL-10r), αTGF-ß, or isotype control antibodies 24 h before each sensitization. The acute allergic skin response, anaphylaxis score, whey-specific IgE, mucosal mast cell protease 1 (mMCP-1), and Treg frequency in the mesenteric lymph nodes (MLNs) and intestinal Foxp3, Il10, and Tgfb mRNA expression were determined. Results: In Whey+GFA mice, intestinal Il10, Tgfb, or Foxp3 mRNA expression was 2-10 times higher (P < 0.05) and the MLN Treg frequency was 25% higher compared with Whey mice (P < 0.05). The acute allergic skin response was 50% lower in Whey+GFA mice compared with Whey mice (P < 0.01), and IL-10 receptor (IL-10r) or TGF-ß neutralizing antibodies prevented this protective effect (P < 0.001). The Whey mice had higher serum mMCP-1 concentrations and whey-immunoglobulin E (-IgE) levels than Sham mice (P < 0.01), whereas these were not higher in Whey+GFA mice, and neutralizing antibodies partially interfered with these responses. Conclusions: Dietary GFAs enhance the Treg frequency in the MLNs and mucosal IL-10 and TGF-ß transcription while suppressing the allergic effector response. Neutralizing antibodies showed that the allergy-protective effect of the GFA diet was mediated by IL-10 and TGF-ß in CMA mice.

Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner.

Intestinal epithelial cells (IEC) drive regulatory T cell (Treg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103+ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC.

Dietary, nondigestible oligosaccharides and Bifidobacterium breve M-16V suppress allergic inflammation in intestine via targeting dendritic cell maturation.

Dietary intervention with short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and Bifidobacterium breve M-16V (Bb) (GF/Bb) suppresses food allergic symptoms in mice, potentially via intestinal epithelial cell (IEC)-derived galectin-9. Furthermore, in vitro studies showed galacto- and fructo-oligosaccharides (GF) to enhance the immunomodulatory capacity of a TLR9 ligand representing bacterial CpG DNA when exposed to IEC. In this study, we investigated whether GF/Bb modulates dendritic cells (DCs) and subsequent Th2 and regulatory T cell (Treg) frequency in the small intestinal lamina propria (SI-LP). BALB/c mice were fed GF/Bb during oral OVA sensitization. DC and T cell phenotype were determined in SI-LP mononuclear cells using flow cytometry. Murine bone marrow-derived DCs (BMDCs) were exposed to recombinant galectin-9 or human monocyte-derived DCs (moDCs) and were cultured in IEC-conditioned medium from GF and TLR9 ligand-exposed HT-29 cells. GF/Bb reduced allergic symptoms and enhanced serum galectin-9 levels, while suppressing activation, restoring phagocytic capacity, and normalizing CD103 expression of SI-LP DCs of OVA-allergic mice. In vitro, galectin-9 suppressed LPS-induced activation markers and cytokine secretion by BMDCs, and IEC-conditioned medium suppressed moDC activation in a galectin-9-dependent manner. Besides suppression of SI-LP DC activation, dietary GF/Bb also lowered the frequency of activated Th2 cells, while enhancing Treg in the SI-LP of OVA-allergic mice compared to the control diet. Dietary intervention with GF/Bb enhances galectin-9 and suppresses allergic symptoms of OVA-allergic mice in association with reduced intestinal DC and Th2 activation and increased Treg frequency in these mice.

Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

UNLABELLED: Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+) (Th1) activated CD69(+)CD4(+) T cells (p<0.001) and reduced percentage of Gata-3(+) (Th2) activated CD69(+)CD4(+)T cells (p<0.001) was detected in the mesenteric lymph nodes (MLN) of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+)Foxp3(+)) could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+) /T-bet(+) (Th1-Tregs) was significantly reduced (p<0.05) in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response. IN CONCLUSION: These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.

The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation.

The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.