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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.

OBJECTIVE: The intestinal microbiota is acknowledged to be essential in brain development and behaviour. Their composition can be modulated by prebiotics such as short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharide (lcFOS). Several studies reported potential health benefit of prebiotics on behaviour. As the prebiotic mixture of scGOS and lcFOS is included in infant formula, we investigated the effects of dietary supplementation with this specific mixture from the day of birth onwards on behaviour and intestinal microbiota development in mice. METHOD: Healthy male BALB/cByJ mice received, from day of birth, a dietary supplement with or without 3% scGOS:lcFOS (9:1). Behavioural tests were performed pre-weaning, in adolescence, early adulthood and adulthood. We assessed faecal microbiota compositions over time, caecal short-chain fatty acids as well as brain mRNA expression of Htr1a, Htr1b and Tph2 and monoamine levels. RESULTS: Compared to control fed mice, scGOS:lcFOS fed mice showed reduced anxiety-like and repetitive behaviour over time and improved social behaviour in adulthood. The serotonergic system in the prefrontal cortex (PFC) and somatosensory cortex (SSC) was affected by the scGOS:lcFOS. In the PFC, mRNA expression of brain-derived neurotrophic factor (Bdnf) was enhanced in scGOS:lcFOS fed mice. Although the bacterial diversity of the intestinal microbiota was unaffected by the scGOS:lcFOS diet, microbiota composition differed between the scGOS:lcFOS and the control fed mice over time. Moreover, an increased saccharolytic and decreased proteolytic fermentation activity were observed in caecum content. DISCUSSION: Supplementing the diet with scGOS:lcFOS from the day of birth is associated with reduced anxiety-like and improved social behaviour during the developmental period and later in life, and modulates the composition and activity of the intestinal microbiota in healthy male BALB/c mice. These data provide further evidence of the potential impact of scGOS:lcFOS on behaviour at several developmental stages throughout life and strengthen the insights in the interplay between the developing intestine and brain.

Beta-lactoglobulin (BLG)-derived peptides may facilitate oral tolerance to whey and prevent cow's milk allergy (CMA). Loading of BLG-peptides in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Pep-NP) may improve this. Here we studied the uptake of NP and the capacity of NP and Pep-NP to activate bone marrow dendritic cells (BMDC). Furthermore, CMA prevention was evaluated by orally exposing three-week-old female C3H/HeOuJ mice to Pep-NP, NP or free peptides (PepMix) for 6 days before oral sensitization with whole whey protein and effects on the spleen and small intestine lamina propria (SI-LP) were studied. In BMDC, NP and Pep-NP enhanced CD40 expression and IL-6 and TNF-α secretion, while tended to decrease CD80 expression and prevented PepMix-induced IL-12 secretion. In vivo, oral exposure to Pep-NP, but not NP or PepMix, prior to whey sensitization tended to partially prevent the acute allergic skin response to whole whey protein. Splenocytes of NP-pre-exposed mice secreted increased levels of whey-specific IL-6, but this was silenced in Pep-NP-pre-exposed mice which also showed reduced TNF-α and IFN-γ secretion. In the SI-LP, Pep-NP pre-exposure reduced the CD4+ T cell frequency in CMA mice compared to PBS pre-exposure. In addition, while NP increased whey-specific IL-6 secretion in the SI-LP, Pep-NP did not and maintained regulatory TGF-β secretion. This study presents a proof-of-concept that PLGA nanoparticles facilitate the capacity of BLG peptides to suppress the allergic response to whole whey protein. Hence, PLGA nanoparticles may be further developed as an adjunct strategy for BLG-peptide-based oral tolerance induction and CMA prevention.

SCOPE: A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side-effects. Non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS), reduced allergy development in murine models. We therefor hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model. METHODS AND RESULTS: After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice were fed a 1% scFOS/lcFOS or control diet and received OIT (1.5 or 15 mg PE). Hereafter, mice were exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE-specific antibody production and numbers of various immune cells. scFOS/lcFOS increased short-chain fatty acid levels in the caecum and reduced the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induced protection against anaphylaxis, whereas 1.5 mg OIT alone did not. OIT, with or without scFOS/lcFOS, induced PE-specific IgG and IgA levels and increased CD103+ dendritic cells in the mesenteric lymph nodes. CONCLUSIONS: scFOS/lcFOS and scFOS/lcFOS combined with a low dose OIT are able to protect against a peanut-allergic anaphylactic response. This article is protected by copyright. All rights reserved.

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.

From Pubmed: " BACKGROUND: Antigen-specific immunotherapy (AIT) is a promising therapeutic approach for both cow's milk allergy (CMA) and peanut allergy (PNA), but needs optimization in terms of efficacy and safety. AIM: Compare oral immunotherapy (OIT) and subcutaneous immunotherapy (SCIT) in murine models for CMA and PNA and determine the dose of allergen needed to effectively modify parameters of allergy. METHODS: Female C3H/HeOuJ mice were sensitized intragastrically (i.g.) to whey or peanut extract with cholera toxin. Mice were treated orally (5 times/week) or subcutaneously (3 times/week) for three consecutive weeks. Hereafter, the acute allergic skin response, anaphylactic shock symptoms and body temperature were measured upon intradermal (i.d.) and intraperitoneal (i.p.) challenge, and mast cell degranulation was measured upon i.g. challenge. Allergen-specific IgE, IgG1 and IgG2a were measured in serum at different time points. Single cell suspensions derived from lymph organs were stimulated with allergen to induce cytokine production and T cell phenotypes were assessed using flow cytometry. RESULTS: Both OIT and SCIT decreased clinically related signs upon challenge in the CMA and PNA model. Interestingly, a rise in allergen-specific IgE was observed during immunotherapy, hereafter, treated mice were protected against the increase in IgE caused by allergen challenge. Allergen-specific IgG1 and IgG2a increased due to both types of AIT. In the CMA model, SCIT and OIT reduced the percentage of activated Th2 cells and increased the percentage of activated Th1 cells in the spleen. OIT increased the percentage of regulatory T cells (Tregs) and activated Th2 cells in the MLN. Th2 cytokines IL-5, IL-13 and IL-10 were reduced after OIT, but not after SCIT. In the PNA model, no differences were observed in percentages of T cell subsets. SCIT induced Th2 cytokines IL-5 and IL-10, whereas OIT had no effect. CONCLUSION: We have shown clinical protection against allergic manifestations after OIT and SCIT in a CMA and PNA model. Although similar allergen-specific antibody patterns were observed, differences in T cell and cytokine responses were shown. Whether these findings are related to a different mechanism of AIT in CMA and PNA needs to be elucidated."

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.

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.