Dietary non-digestible oligosaccharides (NDOs) have a protective effect against allergic manifestations in children at risk. Dietary intervention with NDOs promotes the colonization of beneficial bacteria in the gut and enhances serum galectin-9 levels in mice and atopic children. Next to this, NDOs also directly affect immune cells and low amounts may reach the blood. We investigated whether pre-incubation of whole blood from peanut-allergic patients with NDOs or galectin-9 can affect basophil degranulation.

Dendritic cells (DCs) play an important role in antigen presentation, and are an interesting target for immune-modulation in allergies. Short- and long-chain fructo-oligosaccharides (scFOS/lcFOS, FF) have immunomodulatory capacities, and may influence the outcome of DC antigen presentation.

Proper early life immune development creates a basis for a healthy and resilient immune system, which balances immune tolerance and activation. Deviations in neonatal immune maturation can have life-long effects, such as development of allergic diseases. Evidence suggests that human milk oligosaccharides (HMOS) possess immunomodulatory properties essential for neonatal immune maturation. To understand the immunomodulatory properties of enzymatic or bacterial produced HMOS, the effects of five HMOS (2'FL, 3FL, 3'SL, 6'SL and LNnT), present in human milk have been studied. A PBMC immune model, the IEC barrier model and IEC/PBMC transwell coculture models were used, representing critical steps in mucosal immune development. HMOS were applied to IEC cocultured with activated PBMC. In the presence of CpG, 2'FL and 3FL enhanced IFNγ (p < 0.01), IL10 (p < 0.0001) and galectin-9 (p < 0.001) secretion when added to IEC; 2'FL and 3FL decreased Th2 cell development while 3FL enhanced Treg polarization (p < 0.05). IEC were required for this 3FL mediated Treg polarization, which was not explained by epithelial-derived galectin-9, TGFβ nor retinoic acid secretion. The most pronounced immunomodulatory effects, linking to enhanced type 1 and regulatory mediator secretion, were observed for 2'FL and 3FL. Future studies are needed to further understand the complex interplay between HMO and early life mucosal immune development.

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized β-3'galactosyllactose (β-3'GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or β-3'GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized β-3'GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by β-3'GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized β-3'GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.

Non-digestible oligosaccharides promote colonization of beneficial gut bacteria and have direct immunomodulatory effects. Apical exposure of intestinal epithelial cells (IECs) to short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (scGOS/lcFOS) in a transwell co-culture model enhanced the CpG-induced (TLR-9 ligand) T helper 1 (Th1) phenotype and regulatory IL-10 response of underlying peripheral mononuclear cells (PBMCs) of healthy donors. scGOS is derived from lactose and may pose risks in severe cow's milk allergic patients, and scFOS/lcFOS may be an alternative. The goal of this study was to determine the immunomodulatory effects of scGOS/lcFOS and scFOS/lcFOS in an allergen-specific transwell co-culture model using PBMCs from peanut-allergic patients.

Early life exposure to non-digestible oligosaccharides (NDO) or microbial components is known to affect immune development. NDO in combination with a TLR9 agonist mimicking bacterial triggers (CpG) promoted the secretion of galectins through unknown pathways. We aimed to study the contribution of exosomes in epithelial galectin secretion and subsequent immunoregulation upon exposure to a mixture of NDO by inhibiting exosome biogenesis.

Short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS) and CpG-ODN affect intestinal epithelial cells (IEC). Epithelial IL1α may contribute to allergic sensitization via autocrine mediator release affecting dendritic cells (DC). We studied whether IL1α contributes to Th2-associated mediator release by activated IEC and IEC/DC cocultures and possible modulation by scGOS/lcFOS±CpG-ODN.

Oral tolerance is a promising approach for allergy prevention in early life, but it strongly depends on allergen exposure and proper immune environment. Small tolerance-inducing peptides and dietary immunomodulatory components may comprise an attractive method for allergy prevention in at-risk infants. This study aimed to investigate whether early oral exposure to β-lactoglobulin-derived peptides (BLG-peptides) and a specific synbiotic mixture of short- and long- chain fructo-oligosaccharides (scFOS/lcFOS, FF) and Bifidobacterium breve (Bb) M-16V (FF/Bb) can prevent cow's milk allergy (CMA). Three-week-old female C3H/HeOuJ mice were orally exposed to phosphate buffered saline (PBS), whey protein, or a mixture of four synthetic BLG-peptides combined with a FF/Bb-enriched diet prior to intragastric sensitization with whey protein and cholera toxin. To assess the acute allergic skin response and clinical signs of allergy, mice were challenged intradermally with whole whey protein. Serum immunoglobulins were analyzed after a whey protein oral challenge. Cytokine production by allergen-reactivated splenocytes was measured and changes in T cells subsets in the spleen, mesenteric lymph nodes, and intestinal lamina propria were investigated. Pre-exposing mice to a low dosage of BLG-peptides and a FF/Bb-enriched diet prior to whey protein sensitization resulted in a significant reduction of the acute allergic skin response to whey compared to PBS-pretreated mice fed a control diet. Serum immunoglobulins were not affected, but anaphylactic symptom scores remained low and splenocytes were non-responsive in whey-induced cytokine production. In addition, preservation of the Th1/Th2 balance in the small intestine lamina propria was a hallmark of the mechanism underlying the protective effect of the BLG-peptides-FF/Bb intervention. Prior exposure to BLG-peptides and a FF/Bb-enriched diet is a promising approach for protecting the intestinal Th1/Th2 balance and reducing the allergic response to whole whey protein. Therefore, it might have implications for developing successful nutritional strategies for CMA prevention.

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.

Cow's milk allergy (CMA) prevails in infants and brings increased risk of developing other allergic diseases. Oral administration of specific β-lactoglobulin (BLG)-derived peptides (PepMix) and a specific blend of short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V (FF/Bb) was found to partially prevent CMA development in mice. In this study, we aimed to expand the knowledge on the preventive potential and the underlying mechanisms of this approach. Three-week-old female C3H/HeOuJ mice were orally exposed to PepMix±FF/Bb prior to a 5-week oral sensitization with whole whey and cholera toxin as an adjuvant. The acute allergic skin response was determined after an intradermal challenge with whole whey protein. Following an oral challenge with whey, regulatory T cells (Tregs) in the small intestine lamina propria (SI-LP) and mRNA expression of immune markers in the Peyer's patches (PP) were investigated. The early impact of PepMix and FF/Bb interventions on the immune system during the oral tolerance (OT) induction phase was investigated after the last OT administration. Pre-exposing mice to PepMix+FF/Bb partially prevented the acute allergic skin response compared to PBS and increased Tregs and activated T cells in the SI-LP compared to sham-sensitized mice. It also increased the mRNA expression of Tbet over GATA3 in the PP of whey-sensitized mice. Directly upon the 6-day OT phase, FF/Bb intervention enhanced cecal content levels of propionic and butyric acid in PepMix-fed mice and the former was positively correlated with Foxp3+ cell numbers in the colon. In the PP of PepMix+FF/Bb-exposed mice, IL-22 mRNA expression increased and IL-10 followed the same tendency, while the Foxp3 expression was increased over GATA3 and RorγT. In the colon, the Tbet mRNA expression increased over GATA3, while IL-22 decreased. In addition, the Foxp3+/GATA3+ and regulatory/effector T cell ratios in the mesenteric lymph nodes and the CD11b+/CD11b- conventional dendritic cells ratio in the SI-LP were increased. In conclusion, the FF/Bb diet facilitates the capacity of the specific BLG-peptides to partially prevent the allergic response after sensitization to whole whey protein, possibly by creating a tolerance-prone environment during the OT phase. Such a dietary intervention might contribute to tailoring successful strategies for CMA prevention.

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.

Intestinal epithelial cells (IEC) release immunomodulatory galectins upon exposure to CpG DNA (mimicking bacterial triggers) and short-chain galacto- and long-chain fructo-oligosaccharides (GF). This study aims to investigate the immunomodulatory properties of 2'-fucosyllactose (2'-FL), a non-digestible oligosaccharide (NDO) abundantly present in human milk, using a co-culture model developed to study the crosstalk between IEC and innate and adaptive immune cells. IECs, co-cultured with αCD3/CD28-activated peripheral blood mononuclear cells (PBMC), were apically exposed to NDOs and CpG, washed and co-cultured with immature monocyte-derived dendritic cells (moDC). Subsequently, moDC were co-cultured with naïve CD4+ T-cells. In the presence of CpG, both 2'-FL or GF-exposed IEC enhanced Th1-type IFNγ and regulatory IL-10 secretion of PBMCs, compared to CpG alone, while Th2-type IL-13 was reduced. Both NDOs increased IEC-derived galectin-3, -4, -9 and TGF-β1 of CpG-exposed IEC. Only galectin-9 correlated with all modified immune parameters and TGF-β1 secretion. MoDCs exposed to 2'-FL and CpG-conditioned IEC instructed IFNγ and IL-10 secretion by CD4+ T-cells, suggesting the development of a regulatory Th1 response. These results reveal that 2'-FL and GF could contribute to the mucosal immune development by supporting the effect of microbial CpG DNA associated with the modulation of epithelial galectin and TGF-β1 secretion.

Allergic sensitization starts with epithelial cell activation driving dendritic cells (DCs) to instruct T helper 2 (Th2) cell polarization. Food allergens trigger intestinal epithelial cell (IEC) activation. Human milk oligosaccharides may temper the allergic phenotype by shaping mucosal immune responses.We investigated in vitro mucosal immune development after allergen exposure by combining ovalbumin (OVA)-preexposed IEC with monocyte-derived DCs (OVA-IEC-DCs) and subsequent coculture of OVA-IEC-DCs with Th cells. IECs were additionally preincubated with 2'FL or 3FL.OVA activation increased IEC cytokine secretion. OVA-IEC-DCs instructed both IL13 (p < 0.05) and IFNγ (p < 0.05) secretion from Th cells. 2'FL and 3FL permitted OVA-induced epithelial activation, but 2'FL-OVA-IEC-DCs boosted inflammatory and regulatory T-cell development. 3FL-OVA-IEC lowered IL12p70 and IL23 in DCs and suppressed IL13 (p < 0.005) in T cells, while enhancing IL17 (p < 0.001) and IL10 (p < 0.005).These results show that OVA drives Th2- and Th1-type immune responses via activation of IECs in this model. 2'FL and 3FL differentially affect OVA-IEC-driven immune effects. 2'FL boosted overall T-cell OVA-IEC immunity via DC enhancing inflammatory and regulatory responses. 3FL-OVA-IEC-DCs silenced IL13, shifting the balance towards IL17 and IL10.This model demonstrates the contribution of IEC to OVA Th2-type immunity. 2'FL and 3FL modulate the OVA-induced activation in this novel model to study allergic sensitization.