Linette E. M. Willemsen

Cow Milk Allergy Symptoms Are Reduced in Mice Fed Dietary Synbiotics during Oral Sensitization with Whey

Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. The purpose of this study was to compare effects of dietary supplementation with a prebiotic mixture (Immunofortis), a probiotic strain [Bifidobacterium breve M-16V], or a synbiotic diet combining both on the outcome of the allergic response when provided during oral sensitization with whey in mice. Mice were fed diets containing 2% (wt:wt) Immunofortis and/or the B. breve M-16V (n = 6/group). The acute allergic skin response was determined by measuring ear swelling. Antigen-induced anaphylaxis was scored. Furthermore, whey-specific serum immunoglobulins and mouse mast cell protease-1 (mMCP-1) were determined. In mice fed the synbiotic mixture, the allergic skin response and the anaphylactic reaction were strongly reduced compared with whey-sensitized mice fed the control diet (P < 0.01). Immunofortis or B. breve M-16V alone were significantly less effective in reducing the allergic skin response than the synbiotic diet and did not reduce the anaphylactic reaction. The whey-specific IgE and IgG(1) responses were not affected; however, IgG(2a) was greater in all treated groups than in the control group (P < 0.05). Serum mMCP-1 concentrations, reflecting mucosal mast cell degranulation, were lower in mice fed synbiotics compared with those fed the control diet (P < 0.01). Dietary supplementation with Immunofortis, B. breve M-16V, and particularly the synbiotic mixture, provided during sensitization, reduces the allergic effector response in a murine model of IgE-mediated hypersensitivity that mimics the human route of sensitization. This model shows the potential for dietary intervention with synbiotics in reducing the allergic response to food allergens.

Galectin-9 induced by dietary synbiotics is involved in suppression of allergic symptoms in mice and humans

Prebiotic galacto‐ and fructo‐oligosaccharides (scGOS/lcFOS) resembling non‐digestible oligosaccharides in human milk reduce the development of atopic disorders. However, the underlying mechanisms are still unclear. Galectins are soluble‐type lectins recognizing β‐galactoside containing glycans. Galectin‐9 has been shown to regulate mast cell degranulation and T‐cell differentiation. In this study, the involvement of galectin‐9 as a mechanism by which scGOS/lcFOS in combination with Bifidobacterium breve M‐16V protects against acute allergic symptoms was investigated.

Oligosaccharide-Induced Whey-Specific CD25+ Regulatory T-Cells Are Involved in the Suppression of Cow Milk Allergy in Mice

Dietary intervention with a unique prebiotic nondigestible carbohydrate mixture has been shown to reduce the development of allergic disease in infants at risk. In this study, the involvement of CD25(+) regulatory T-cells (Treg) in the carbohydrate-induced effects was investigated in mice orally sensitized with whey using adoptive transfer experiments. Donor mice were sensitized with whey and fed a diet containing short-chain galacto-, long-chain fructo- and acidic-oligosaccharides, or a control diet starting 2 wk before sensitization. The acute allergic skin reaction upon intradermal whey challenge was determined and whey-specific Ig were measured. Splenocytes of the donor mice were transferred to naïve recipient mice after partial ex vivo depletion of CD25(+) Treg. The prebiotic diet clearly diminished the acute allergic skin reaction (P < 0.001). Whey-sensitized recipient mice transferred with splenocytes from whey-sensitized, prebiotic-fed donor mice displayed almost complete prevention of the acute allergic skin reaction compared with mice receiving cells from sham-sensitized, prebiotic-fed donor mice (P < 0.001). Partial depletion of CD25(+) T-cells inhibited these effects (P < 0.001), although IgE sensitization was not prevented. This study indicates the involvement of whey-specific CD25(+) Treg in the suppression of the allergic effector response induced by dietary intervention with prebiotics.

Polyunsaturated fatty acids support epithelial barrier integrity and reduce IL-4 mediated permeability in vitro

BackgroundThe intestinal mucosa functions as a barrier against harmful dietary and microbial antigens. An intact gut barrier forms a prerequisite for protection against infection and allergy. Both allergic and inflammatory mediators (e.g. IL-4, IFN-γ) are known to compromise the epithelial barrier integrity by enhancing permeability. Breast milk provides protection against infection and allergy and contains polyunsaturated fatty acids (PUFA).Aim of the studyAlthough PUFA are commonly used in infant formulas their effect on intestinal barrier is still poorly understood. Therefore the effects of distinct PUFA (n-6: LA, GLA, DGLA, AA; n-3: ALA, EPA, DHA) and a fat blend with PUFA composition similar to that of the human breast milk fat fraction, on barrier integrity were investigated.MethodsHuman intestinal epithelial cells (T84) were pre-incubated with individual PUFA or a lipase treated fat blend, with or without subsequent IL-4 exposure. Barrier integrity was evaluated by measuring transepithelial resistance and permeability. Membrane phospholipid composition was determined by capillary gas chromatography.ResultsDGLA, AA, EPA, DHA and to a lesser extend GLA enhanced basal TER and strongly reduced IL-4 mediated permeability, while LA and ALA were ineffective. Furthermore, the lipase treated fat blend effectively supported barrier function. PUFA were incorporated in the membrane phospholipid fraction of T84 cells.ConclusionsLong chain PUFA DGLA, AA, EPA and DHA were particularly effective in supporting barrier integrity by improving resistance and reducing IL-4 mediated permeability. Fat blends that release specific PUFA upon digestion in the gastrointestinal tract may support natural resistance.

Glycan recognition at the interface of the intestinal immune system: target for immune modulation via dietary components.

The intestinal mucosa is constantly exposed to the luminal content, which includes micro-organisms and dietary components. Prebiotic non-digestible oligosaccharides may be supplemented to the diet to exert modulation of immune responses in the intestine. Short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS), functionally mimicking oligosaccharides present in human milk, have been reported to reduce the development of allergy through modulation of the intestinal microbiota and immune system. Nonetheless, the underlying working mechanisms of scGOS/lcFOS are unclear. Intestinal epithelial cells lining the mucosa are known to express carbohydrate (glycan)-binding receptors that may be involved in modulation of the mucosal immune response. This review aims to provide an overview of glycan-binding receptors, in particular galectins, which are expressed by intestinal epithelial cells and immune cells. In addition, their involvement in health and disease will be addressed, especially in food allergy and inflammatory bowel disease, diseases originating from the gastro-intestinal tract. Insight in the recognition of glycans in the intestinal tract may open new avenues for the treatment of intestinal inflammatory diseases by either nutritional concepts or pharmacological intervention.

Contribution of IgE and immunoglobulin free light chain in the allergic reaction to cow's milk proteins

BACKGROUND Cows milk allergy (CMA) affects 2.5% of young infants. In previous murine studies it was observed that allergic sensitization to the major cows milk allergens casein and whey led, respectively, to IgE-independent and IgE-dependent clinical responses. OBJECTIVES In this study the involvement of immunoglobulin free light chains (Ig-fLCs) in the hypersensitivity response to cows milk proteins was explored in mice, and Ig-fLC serum levels were determined in children affected by CMA or atopic dermatitis (AD). METHODS Mice were orally sham, casein, or whey sensitized. Acute allergen-specific skin responses were determined, and serum immunoglobulin and Ig-fLC concentrations were measured. Ig-fLC dependency was validated by using the Ig-fLC blocker F991 in actively and passively sensitized mice. Ig-fLC serum concentrations were measured in a cohort of infants with CMA and infants with AD. RESULTS After sensitization, no specific IgE was detectable in sera of casein-sensitized mice, whereas specific IgE levels were enhanced in whey-sensitized mice. Instead, Ig-fLC levels were increased in sera from casein-sensitized mice. Furthermore, blocking Ig-fLCs strongly diminished the allergic skin responses not only in casein-sensitized mice but also in mice transferred with splenocyte supernatants of casein-sensitized mice. In both patients with CMA and patients with AD, serum Ig-fLC concentrations were significantly enhanced. CONCLUSIONS This study indicates that sensitization with cows milk proteins can lead to both IgE-dependent and Ig-fLC-dependent allergic hypersensitivity responses. Also, in children affected with CMA or AD, serum Ig-fLC concentrations were increased, implying the relevance of Ig-fLC measurements in the diagnoses of human allergic disease.

Dietary long chain n‐3 polyunsaturated fatty acids prevent allergic sensitization to cow's milk protein in mice

Cows milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease.

Oral tolerance induction by partially hydrolyzed whey protein in mice is associated with enhanced numbers of Foxp3+ regulatory T-cells in the mesenteric lymph nodes.

To cite this article: van Esch BCAM, Schouten B, de Kivit S, Hofman GA, Knippels LMJ, Willemsen LEM, Garssen J. Oral tolerance induction by partially hydrolyzed whey protein in mice is associated with enhanced numbers of Foxp3+ regulatory T‐cells in the mesenteric lymph nodes. Pediatr Allergy Immunol 2011: 22: 820–826.

Acute Allergic Skin Reactions and Intestinal Contractility Changes in Mice Orally Sensitized against Casein or Whey

Background: Cow’s milk allergy (CMA) is characterized by hypersensitivity against casein or whey, affecting 2.5% of young infants. The pathogenesis of CMA involves IgE as well as non-IgE-mediated reactions and clinical symptoms are found in the skin, lungs and gastrointestinal tract. In this study, local and systemic immunopathology was determined in whey- or casein-allergic mice. Methods: Mice were orally sensitized with casein or whey using cholera toxin as an adjuvant. Serum immunoglobulins and the acute allergic skin reaction (ear swelling 1 h after intradermal allergen challenge) were determined to reveal systemic hypersensitivity. Furthermore, pathophysiological changes were assessed within the intestine. Results: An acute allergic skin reaction was induced in both whey- and casein-sensitized mice. In these mice, whey-specific IgE, IgG1, IgG2a and casein-specific IgG1 levels were found to be increased. In addition, the serum mouse mast cell protease-1 (mMCP-1) concentration was enhanced, reflecting mast cell degranulation. Indeed, the number of mMCP-1-positive mast cells within the colon was diminished in both whey- and casein-sensitized mice. Only in casein-sensitized mice isometric contraction of the colon was reduced, reflecting motility alterations. Conclusion: Mice, orally sensitized against casein or whey, revealed an allergen-specific acute allergic skin reaction. In casein-sensitized mice, hypocontractility of the colon reflected pathophysiological changes within the intestine. Allergen-induced ear swelling and intestinal contractility changes are novel parameters in animal models of CMA which may add to the search for new therapeutic strategies to relieve symptoms of CMA.

Apical TLR ligation of intestinal epithelial cells drives a Th1-polarized regulatory or inflammatory type effector response in vitro.

Intestinal epithelial cells (IEC) separate the mucosal immune system from the external milieu. Under inflammatory conditions, Toll-like receptor (TLR) expression by IEC is increased. In a transwell co-culture model immune modulation by IEC upon TLR ligation was studied. Human IEC (HT-29 and T84) grown on filters were apically or basolaterally exposed to TLR4 or TLR9 ligands and co-cultured with CD3/CD28-activated healthy donor PBMC in the basolateral compartment. TLR4 ligation of IEC (HT-29) enhanced the production of TNF-α and IEC-derived MDC and decreased numbers of Foxp3(+) regulatory T cells. Neutralization of TSLP abrogated TLR4-induced TNF-α secretion. In contrast, apical TLR9 ligation of IEC (HT-29 and T84) enhanced IFN-γ and IL-10 secretion and increased the number of activated T(h)1 cells. The increase in IFN-γ secretion depended on the presence of IEC. Furthermore, CD14 expression on monocytes was reduced coinciding with enhanced intracellular IL-10 and decreased TNF-α production. However, basolateral TLR9 ligand exposure of HT-29 cells resulted in enhanced IFN-γ, IL-6 and TNF-α, while IL-10 secretion remained unaltered. TLR4 and TLR9 ligands reduced IL-13 secretion in presence and absence of apically exposed IEC and enhanced IL-12 secretion in presence of IEC. These data suggest that TLR4 ligation of IEC drives an inflammatory, while apical TLR9 ligation drives a regulatory T(h)1 effector immune response in vitro in a polarized manner. IEC may be important modulators of the mucosal effector immune response.