Betty C. A. M. van Esch

1 alpha,25-dihydroxyvitamin D(3) potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: Role for IL-10 and TGF-beta

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a potent inhibitor of NF-κB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)2D3 potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-β are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 μg. Interestingly, coadministration of 10 ng of 1,25(OH)2D3 with 100 μg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-β and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-κB pathway using the selective IκB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-β before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)2D3 potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-β play a crucial role in the effector phase of this mouse model.

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.

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.

Mechanisms underlying immune effects of dietary oligosaccharides

The WHO refers to human milk as the nutritional gold standard for term infants. Human milk contains many immunomodulatory compounds, including oligosaccharides. Human-milk oligosaccharides can serve as prebiotics because the nondigestible oligosaccharides present in human milk show a clear bifidogenic effect on the gut microbiota. Dietary oligosaccharide structures that have prebiotic effects similar to human-milk oligosaccharides include galacto-oligosaccharides, fructo-oligosaccharides, and pectin-derived acidic oligosaccharides. Both animal studies and human clinical trials showed that dietary intervention with these dietary oligosaccharides in early life could lead to the prevention of atopic dermatitis, food allergy, and allergic asthma. The immune-modulating effects of these oligosaccharides are likely assisted via alteration of the intestinal microbiota or in a microbiota-independent manner by direct interaction on immune cells or both. In this review, an overview of the prebiotic role of dietary oligosaccharides on the microbiota and the microbiota-independent immune modulation by these prebiotics is provided. In addition, recent publications that report on the pathways by which the oligosaccharides might exert their direct immunomodulatory effect are summarized.

Indoleamine 2,3-dioxygenase–dependent tryptophan metabolites contribute to tolerance induction during allergen immunotherapy in a mouse model

BACKGROUND The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. OBJECTIVE We examined (1) whether IDO activity is required during tolerance induction by allergen immunotherapy or for the subsequent suppressive effects on asthma manifestations and (2) whether tryptophan depletion or generation of its downstream metabolites is involved. METHODS Ovalbumin (OVA)-sensitized and OVA-challenged BALB/c mice that display increased airway responsiveness to methacholine, serum OVA-specific IgE levels, bronchoalveolar eosinophilia, and TH2 cytokine levels were used as a model of allergic asthma. Sensitized mice received subcutaneous optimal (1 mg) or suboptimal (100 microg) OVA immunotherapy. RESULTS Inhibition of IDO by 1-methyl-DL-tryptophan during immunotherapy, but not during inhalation challenge, partially reversed the suppressive effects of immunotherapy on airway eosinophilia and TH2 cytokine levels, whereas airway hyperresponsiveness and serum OVA-specific IgE levels remained suppressed. Administration of tryptophan during immunotherapy failed to abrogate its beneficial effects toward allergic airway inflammation. Interestingly, administration of tryptophan or its metabolites, kynurenine, 3-hydroxykynurenine, and xanthurenic acid, but not 3-hydroxyanthranilinic acid, quinolinic acid, and kynurenic acid, during suboptimal immunotherapy potentiated the reduction of eosinophilia. These effects coincided with reduced TH2 cytokine levels in bronchoalveolar lavage fluid, but no effects on IgE levels were detected. CONCLUSION During immunotherapy, the tryptophan metabolites kynurenine, 3-hydroxykynurenine, and xanthurenic acid generated through IDO contribute to tolerance induction regarding TH2-dependent allergic airway inflammation.

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.

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.

Regulatory T-cells have a prominent role in the immune modulated vaccine response by specific oligosaccharides

Regulatory T-cells are increasingly important in vaccine strategies. In a Flu-vaccination model the role of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) and the immune modulation by orally supplied prebiotic oligosaccharides consisting of scGOS/lcFOS/pAOS, were assessed using anti-CD25 (PC61) mediated depletion studies. As expected, in C57BL/6J mice the Flu-vaccination resulted in significantly (p<0.001) increased DTH responses when receiving scGOS/lcFOS/pAOS. In addition, increased T-bet expression of activated CD4(+) T-cells was detected compared to placebo. In vivo depletion of CD25(+) Tregs significantly (p<0.05) increased basal DTH responses, indicating the suppressive function of these CD25(+) Tregs normally present. Surprisingly, in vivo Tregs depletion diminished scGOS/lcFOS/pAOS induced immune modulation completely to control levels (p<0.05). Although no difference in number, percentage or activation of Tregs could be determined after scGOS/lcFOS/pAOS supplementation, changes in Treg function still remains to be investigated. In conclusion, CD25(+) Tregs have an important role in modulated Flu-vaccine responses induced by scGOS/lcFOS/pAOS.

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.

Non-digestible oligosaccharides reduce immunoglobulin free light-chain concentrations in infants at risk for allergy.

To cite this article: Schouten B, Van Esch BCAM, Kormelink TG, Moro GE, Arslanoglu S, Boehm G, Knippels LMJ, Redegeld FA, Willemsen LEM, Garssen J. Non‐digestible oligosaccharides reduce immunoglobulin free light‐chain concentrations in infants at risk for allergy. Pediatr Allergy Immunol 2011; 22: 537–542.