Original Research published: 12 January 2017 doi: 10.3389/fimmu.2016.00673
A Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates Partial Non-Responsiveness to Whey Protein in Mice Orally Exposed to β-Lactoglobulin-Derived Peptides Atanaska I. Kostadinova1,2, Laura A. P. M. Meulenbroek2, Betty C. A. M. van Esch1,2, Gerard A. Hofman1, Johan Garssen1,2, Linette E. M. Willemsen1 and Léon M. J. Knippels1,2* Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands, Immunology, Nutricia Research, Utrecht, Netherlands
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Edited by: Rachel R. Caspi, National Institutes of Health, USA Reviewed by: Sin-Hyeog Im, Pohang University of Science and Technology, South Korea Javier Ochoa-Reparaz, Eastern Washington University, USA *Correspondence: Léon M. J. Knippels [emailprotected] Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology Received: 04October2016 Accepted: 20December2016 Published: 12January2017 Citation: KostadinovaAI, MeulenbroekLAPM, vanEschBCAM, HofmanGA, GarssenJ, WillemsenLEM and KnippelsLMJ (2017) A Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates Partial Non-Responsiveness to Whey Protein in Mice Orally Exposed to β-Lactoglobulin-Derived Peptides. Front. Immunol. 7:673. doi: 10.3389/fimmu.2016.00673
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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 Abbreviations: BLG, β-lactoglobulin; CMA, cow’s milk allergy; CT, cholera toxin; ELISA, enzyme-linked immunosorbent assay; FF/Bb, specific synbiotic mixture of short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V; MLN, mesenteric lymph nodes; PBS, phosphate buffered saline; PepMix, mixture of four BLG-derived peptides; SCFA, shortchain fatty acids; Th, T helper cells; Treg, regulatory T cells.
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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. Keywords: acute allergic skin response, dietary interventions, food allergy, mouse models, oral tolerance, peptides, prevention, T cells
INTRODUCTION
gut microbiota, resembling the situation in breast-fed infants (16–19). Bacterial dysbiosis, especially abnormal levels of bifidobacteria, has been associated with increased risk of allergy (20, 21). Interestingly, non-digestible oligosaccharides, such as inulin and other fructo-oligosaccharides, are selectively utilized by bifidobacteria in both rats and humans (22) and increase gut bifidobacteria by 10-fold in healthy volunteers (23). Studies in rodents suggest the involvement of T helper 1 (Th1) and T regulatory (Treg) cells in the prevention of allergic asthma and cow’s milk allergic symptoms by dietary non-digestible oligosaccharides (24, 25). Combining fructo-oligosaccharides with a probiotic bacterial strain such as Bifidobacterium breve (Bb) M-16V (a concept known as synbiotics) might result in stronger immunomodulatory properties. The M-16V bacterial strain is known for its anti-allergic activity (26, 27) and has shown beneficial effects in infants with atopic dermatitis (28). Further, the use of synbiotic mixtures containing Bb M-16V was found effective in preventing CMA allergic symptoms (29, 30) as well as house dust mite-induced allergic asthma in mice (31). In a recent clinical trial with infant formulas, it was reported that children fed a formula containing fructo-oligosaccharides and Bb M-16V had an increased percentage bifidobacteria in their feces (32). Bb M-16V is suggested to enhance the homing process of naïve T cells to mesenteric lymph nodes (MLN), induce mucosal IgA production (33), and upregulate the TGF-β1 signaling (34), while they can partially modulate the TNF-α signaling in epithelial cells (35). Therefore, a dietary synbiotic mixture combining fructo-oligosaccharides and Bb M-16V is of interest for providing generic immunomodulation in preventive strategies for food allergy. In this study, we assessed the potential of an early oral exposure to a mixture of four synthetic BLG-derived peptides and a specific mixture of short- and long-chain fructo-oligosaccharides (scFOS/lcFOS, FF) and Bb M-16V (FF/Bb) to prevent allergy development in a murine model of orally induced CMA. We hypothesize that providing the right immune environment by means of a synbiotic diet during BLG-peptide presentation to immune cells would improve the capacity of the peptides to prevent allergic symptoms to the whole whey protein.
Food allergies are becoming a serious health concern worldwide. Cow’s milk allergy (CMA) is the most prevalent food allergy in infancy and the earliest to occur. Even though the majority of cow’s milk allergic infants outgrow their allergy (1), they are often affected by allergies to other foods, such as peanut; a phenomenon referred to as the “Food Allergic March” (2). CMA further plays an important role in the atopic march, meaning that early life CMA may predispose to inhalant-triggered hypersensitivity or asthma later in life (3). Food allergy is suggested to occur due to defective oral tolerance (4). Oral tolerance is the process that actively suppresses the immune response to ingested harmless antigens (5, 6). Research is, therefore, focused on actively inducing or repairing oral tolerance. Studies in mice document that oral tolerance is a strongly antigen-dependent process (7), emphasizing the importance of antigen exposure in early life. Recent reports reveal that consumption of peanuts by Jewish infants in Israel is associated with fewer occurrences of peanut allergy (8) and that feeding at-risk infants the allergenic food early in life can prevent food allergy development (9). However, exposure to the intact antigen might also result in sensitization or allergic symptoms development in infants at high risk of developing food allergy. To avoid this, oral tolerance approaches implementing allergen fragments can be used instead. For instance, partially hydrolyzed whey proteins were shown to suppress allergic symptoms to cow’s milk in mice (10). Even small immunogenic peptides containing T cell epitopes can be potent inducers of tolerance and may be a suitable alternative to whole protein antigens (11). Supplementing hydrolyzed formula with specific peptides was found effective in preventing the allergic response to the native protein (12). In follow-up studies, a reduced allergic sensitization to the major cow’s milk protein β-lactoglobulin (BLG) was observed when it was coadministered with BLG-derived peptides (13). Meulenbroek etal. further reported that oral pre-exposure to synthetic BLG-derived peptides reduces the allergic skin response to whole whey protein (14). Altogether, these studies support the hypothesis that specific protein fragments may be suitable for allergen-specific immunomodulation. Generic immunomodulation suggests the use of dietary components with beneficial immunomodulatory properties to support immune system maturation and natural oral tolerance development by providing the right environment for oral tolerance induction (15). Supplementing infant formulas with dietary prebiotics reduces the prevalence of atopic manifestations and induces bifidobacteria- and lactobacilli-predominating
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MATERIALS AND METHODS Peptides
Four 18-AA-long peptides from the B variant of BLG were synthetically produced by JPT Peptide Technologies (Berlin, Germany). The four peptides contain a 12-AA-long overlap
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Oral Tolerance Induction, Sensitization, and Challenge of Mice
(Figure1) and were previously screened in human T cell lines and used in an animal model (14). The BLG-derived peptides were dissolved in sterile phosphate buffered saline (PBS; Lonza, Walkerville, MD, USA) and mixed until each peptide was at a concentration of 0.08mg/mL (further referred to as PepMix).
Upon arrival, mice were fed the control or the FF/Bb diet adlibitum for a period of 9 days (Figure2). In the same week, mice were orally exposed (using a blunt needle) to 0.16 mg PepMix (0.04 mg of each BLG-derived peptide), 50 mg whey protein (DMV International, Veghel, The Netherlands), or PBS (0.5mL) daily for 6days. From day 0, all mice were maintained on control diet and sensitized as previously described (10). Five days after the last sensitization, an intradermal challenge was performed to assess the allergic response. Mice were challenged orally with 50mg whey, followed by blood sampling at 2h and blood sampling and sacrifice at 18 h after oral challenge. Serum was obtained and stored at −80°C until measurement.
Diets
Semi-purified cow’s milk protein-free standard mouse chow was composed according to a AIN-93G recipe (control diet) and supplemented with 1% (wt:wt) of non-digestible scFOS (Raftilose P95, Beneo Orafti S.A., Oreye, Belgium) and lcFOS (Raftiline HP, Beneo Orafti S.A.) in a ratio 9:1 and 2% (wt:wt) 2×109CFU/g Bb M-16V (Morinaga Milk Industry, Tokyo, Japan) (FF/Bb diet; Research Diet Services, Wijk bij Duurstede, The Netherlands). The synbiotic components were mixed through the diet and pressed into pellets. Diets were stored at 4°C prior to use.
Evaluation of the Allergic Response
To measure the acute allergic skin response, mice were challenged intradermally in the ear pinnae with 10µg whey protein per ear. Ear thickness was recorded before and 1h after the intradermal challenge using a digital micrometer (Mitutoyo, Veenendaal, The Netherlands) and the acute skin response was calculated as Δ=ear thickness at 1h−basal ear thickness and is expressed as delta micrometer. The anaphylactic shock symptoms were scored according to a previously described table (29).
Animals
Three-week-old pathogen-free female C3H/HeOuJ mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Mice were housed in the animal facility of Utrecht University. This study was carried out in accordance with the recommendations of the Animal Ethics Committee of Utrecht University. The protocol was approved by the Animal Ethics Committee of Utrecht University (approval number DEC2014.II.12.100).
Allergen-Specific Immunoglobulins in Serum
Serum whey- and BLG-specific immunoglobulins were quantified by means of an enzyme-linked immunosorbent assay (ELISA) as previously described (10), with few modifications. Briefly, high binding Costar 9018 plates (Corning Inc., New York, NY, USA) were coated with 20 µg/mL whey or BLG protein in carbonate–bicarbonate coating buffer (Sigma-Aldrich, Zwijdrecht, The Netherlands) overnight at 4°C. Plates were washed, blocked for 1 h with 0.5% bovine serum albumin (Sigma-Aldrich)/0.05% Tween-20 (Merck, Billerica, MA, USA) buffer, and serum samples
FIGURE 1 | Sequence information of the four synthetic peptides.
FIGURE 2 | A schematic overview of the murine model for cow’s milk allergy prevention. CT, cholera toxin.
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were incubated for 2h at room temperature. After washing, 1µg/ mL of biotin-labeled rat anti-mouse IgE, IgG1, or IgG2a detection antibody (BD Biosciences, San Jose, CA, USA) was incubated for 1.5 h. The plates were washed and incubated for 1 h with streptavidin-horse radish peroxidase (Sanquin, Amsterdam, The Netherlands), washed again and the reaction was developed with 3,3′,5,5′-tetramethylbenzidine (TMB, Thermo Fisher Scientific, Waltham, MA, USA). The reaction was stopped with 2M H2SO4, and absorbance was measured at 450nm on a Benchmark plate reader (Bio-Rad, Veenendaal, The Netherlands).
(eBioscience). For detecting transcription factors, cells were first fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) according to manufacturer’s protocol and then stained with Foxp3-APC and RorγT-PE antibodies (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia).
Ex Vivo Restimulation Assay and Cytokine Production
Spleens were removed aseptically and single cell suspensions were obtained as described above. Splenocytes (6×105) were cultured either with medium or with 500µg/mL whey protein for 5days at 37°C, 5% CO2. Supernatants were collected and analyzed for IL-5, IL-13, IL-10, IL-17A, and IFN-γ by means of a Cytometric Bead Array Flex Set assay (BD Biosciences) following manufacturer’s instructions. Results were obtained with BD FACSCanto II flow cytometer and analyzed with FCAP v.3.0 software (Becton Dickinson).
Short-Chain Fatty Acid (SCFA) Concentrations in Cecum
Cecal content was collected and immediately frozen in liquid nitrogen. Samples were stored at −80°C until measurement. Concentration of acetic, propionic, and butyric acids were determined by means of gas chromatography as previously described (36), using 2-ethylbutyric acid as internal standard.
Cell Isolation from Tissues
Statistical Analysis
Lymphocytes were isolated from spleen, MLN, and small intestine lamina propria. Spleens and MLN were crushed through 70µm cell strainers. Splenocyte suspension was incubated with lysis buffer (8.3g NH4Cl, 1g KHC3O, and 37.2mg EDTA dissolved in 1L demi water and filter sterilized) to remove red blood cells and then resuspended in RPMI 1640, 10% fetal bovine serum and penicillin (100 U/mL)/streptomycin (100 µg/mL). For the isolation of the lamina propria cells, the small intestine was removed, cleared of Peyer’s patches, washed in cold PBS, opened longitudinally, and minced in 0.5-cm fragments. Samples were washed in Hank’s Balanced Salt Solution (HBSS; Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 15 mM HEPES (Gibco, Life Technologies, Carlsbad, CA, USA), pH=7.2 followed by 4 × 15-min incubations with HBSS supplemented with 15mM HEPES, 5mM Na2-EDTA, 10% fetal bovine serum, and penicillin (100U/mL)/streptomycin (100µg/mL), pH=7.2. The fragments were washed in RPMI 1640, 5% fetal bovine serum and penicillin (100 U/mL)/streptomycin (100µg/mL) and incubated 2 × 45min with an enzyme solution containing 0.25mg/mL Collgenase type VIII (Sigma-Aldrich). To collect the lamina propria cells, fragments were vortexed for 10s after each incubation and passed through a 70µm cell strainer.
For all statistical analyses, GraphPad Prism 6.0c software for Macintosh (GraphPad Software, San Diego, CA, USA) was used. Anaphylactic shock scores and serum immunoglobulins were analyzed by Kruskal–Wallis test followed by Dunn’s posthoc test for seven pre-selected comparisons. All other data were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison posthoc test for selected groups. SCFA data and splenocytes cytokine results were first LOG-transformed. For testing correlations non-parametric Spearman correlation coefficient test was used. All data are presented as mean±SEM of 4–8 animals per group. p
