Attenuation of Allergic Immune Response Phenotype by

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Attenuation of Allergic Immune Response Phenotype by Mannosylated Egg White in Orally Induced Allergy in Balb/c Mice Prithy Rupa,† Soichiro Nakamura,§ Shigeru Katayama,§ and Yoshinori Mine*,† †

Department of Food Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario N1G 2W1, Canada Food Biotechnology Laboratory, Graduate School of Agricultural Science, Shinshu University, Nagano, Japan

§

ABSTRACT: Food allergies are attributed to an imbalance in immune response to ubiquitous antigens. A previous study demonstrated that mannose glycation (mannosylation) of ovalbumin decreased allergenicity in vivo. The proposed research targets mannosylation of various common allergens that may help prevent food allergy. Balb/c mice (n = 8) were sensitized toxin egg white, peanut, and whey and treated with mannosylated forms of the test antigens. Glucosylated peanut and cholera toxins were used as controls. Allergic status was assessed as clinical signs, serum histamine, mouse mast cell protease (MMCP), antibody activity, cytokines, and T regulatory cells (T-regs). Significant preventative effects were observed with mannosylated egg white treatment such as reduced clinical signs, histamine, MMCP, specific G, G1, and E antibody activities, and IL-4 and increased IL-10 and CD25+ Foxp3+ cells. Other groups did not differ significantly. It was concluded that mannosylated egg white provides a powerful tool to prevent allergic phenotypes with possible relevance to control human egg allergy. KEYWORDS: mannosylation, egg white, peanut, whey, immune tolerance, food allergy



INTRODUCTION Food is a major determinant of chronic diseases such as allergies. Food allergy is a major health problem that affects about 4% of the adult population and 6−8% of children in North America and is attributed to a dysregulation in immune response (IR).1 Predisposition to type-2 IR associated with allergies is on the increase, and treatments include strategies that may alter the immune phenotype to a biased type-1 response. Egg, peanut, and milk allergies are among the eight most common food allergies in humans, and some children become tolerant against egg by the age of 5.2 However, egg allergy is also known to persist in others, and the onset of egg allergy is known to continue through adulthood, similar to peanut and milk allergies.3 In keeping with the widespread practice of allergy medicine, avoidance of exposure has been the main remedial action. Molecular and cellular approaches to study in vivo adaptive and/or innate IRs with allergy as a phenotype of immune variation are important to determine alternative management methods to cure allergies. Oral immunotherapy is now increasingly used to treat food allergies.4 Strategies for inducing oral tolerance have been proposed, including the use of antigen-specific hypoallergenic products with extensively reduced allergenicity. Various strategies for masking food protein allergenicity have been reported such as heat treatment, fermentation, enzymatic hydrolysis, genetic modification, and sugar conjugation. Of all these processes, conjugation with reducing sugars through the Maillard reaction seems to be a promising and safe method for masking food protein allergenicity. The Maillard reaction can change the functional properties of the antigen under controlled dry-heating conditions.5 This may be due to the change in the structure of the allergens, thereby the epitopes being made less available to antibody receptors. The influence of glycans on antigen processing and T cell recognition has particular relevance to the induction of tolerance to self© 2014 American Chemical Society

antigens. Controversial data exist with regard to effects of Maillard reaction and glycation on allergenicity. It was recently shown that glycation with glucose in conjunction with thermal denaturation led to a decrease of IgE binding capacity of peanut Ara2/Ara6 proteins;6 however, glycation preserved more mediator-releasing capacity of the proteins compared with heating alone. In addition, moderate heat treatment and glycation of β-lactoglobulin did not have a drastic influence on the recognition of the protein by IgE.7 Glycated ovalbumin (OVA) was found to be more allergenic than native OVA,8 and glucose-conjugated OVA had enhanced CD4 + T cell activation.9 A recent paper showed that pyyraline, a glycation structure, enhanced the potential allergenicity of OVA.10 However, research performed here recently has demonstrated that ovalbumin (OVA) conjugated with mannose and not glucose alleviated orally induced egg allergy in mice.11 Mannose receptors (MR) on dendritic cells are known to recognize a wide range of both endogenous and exogenous ligands through their carbohydrate moieties such as mannose,12,13 suggesting a major link between glycosylation pattern and allergen recognition. The role of mannosylation in allergen recognition was investigated recently, and it was concluded that mannan seems to be the dominant sugar moiety associated with allergens. This was consistent with MR being the main receptor involved in allergen recognition and uptake by DCs.14 We have recently demonstrated that mannose-conjugated ovalbumin (OVA) does trigger the dendritic cells (DCs) with reduced maturation and uptake. This played a vital role in downstream cytokine and IgE production via decreased activation of CD4+ T cells.11 There is a substantial body of evidence from our Received: Revised: Accepted: Published: 9479

June 30, 2014 September 3, 2014 September 12, 2014 September 12, 2014 dx.doi.org/10.1021/jf503109r | J. Agric. Food Chem. 2014, 62, 9479−9487

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Figure 1. Experimental design. Mice were sensitized with 1 mg of egg white (EW), peanut (PN), and whey proteins and 10 μg of cholera toxin (CT) for 4 weeks, twice a week, followed by a 4 week treatment with mannosylated EW, PN, and whey proteins. The glucosylated PN group was used as a positive test group, and the negative control group received PBS. Clinical signs of allergy were recorded post oral challenge (0−30 min), and all mice were euthuanized for collection of blood and spleen. Both blood and spleen were pooled within groups such that n = 4. nonmodified form. All samples were dialyzed (5 kDa cutoff, Millipore, Bedford, MA, USA) with PBS to get rid of the nonconjugated saccharides. As an additional test group, PN proteins were glycated with glucose to be used as a positive control, on the basis of our earlier results that OVA conjugated to glucose did not alleviate allergic signs in mice.11 Determination of Molar Binding Ratios. Total carbohydrate (CHO) content was determined by the phenol/sulfuric acid colorimetric method at 490 nm with D-mannose as standard.18 Protein concentration was measured using the standard bicinchoninic acid method.19 Due to complex total proteins used, conjugation binding ratios for binding of CHO to total protein were calculated on the basis of the concentration using the following formula: concentration of CHO/concentration of total protein. SDS-PAGE. Glycation was also confirmed with running the conjugated samples on sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) gel. Each of the samples was mixed with 2× sample loading buffer and incubated for 10 min at 65 °C prior to SDS-PAGE on a 12% polyacrylamide separating gel with a 5% stacking gel. Electrophoresis was performed at a constant current of 30 mA using a mini Protean apparatus (Bio-Rad). The gel was placed into Coomassie Blue stain solution for 1 h postelectrophoresis and then destained in distilled water for 1 h and photographed. Mice and Sensitization. All animal use protocols were based on Canadian Council for Animal Care guidelines and approved by the University of Guelph Animal Care Committee (eAUP No-1567). Briefly, 64 female Balb/c mice were randomly divided into eight groups (n = 8/group). Mice were orally sensitized with 1 mg of EW, PN, and whey proteins adjuvanated with 10 μg of cholera toxin (CT;

earlier research that mannosylation of OVA did have a potential impact on DCs and antigen recognition and uptake and in reducing allergenicity in vivo in mice. With this background we proposed to study different mannosylated glycoforms of major food allergens such as egg white (EW), peanut (PN), and whey proteins on tolerance induction in vivo in mice. It would be of interest to know under “the glycation allergen concept” if mannosylation influences different allergens in a similar fashion as observed with OVA with regard to reducing allergy or otherwise.



MATERIALS AND METHODS

Chemicals. Carbohydrates such as D-mannose and D-glucose were obtained from Sigma (Oakville, ON, Canada). All antibodies used in the experiments were purchased from BD Biosciences (Pharmingen, San Diego, CA, USA), Sigma (Sigma-Aldrich, Oakville, ON, Canada), or eBioscience (eBiosciences, San Diego, CA, USA). Glycation of Common Allergens. Egg white, PN, and whey proteins were all in-house-extracted and partially purified according to methods previously described.15−17 Maillard reaction or glycation was performed as described in a previously established method with slight modifications.11 In brief, mannose (Sigma-Aldrich, St. Louis, MO, USA) was added to the three proteins at a ratio of 1:1 (w/w protein/ mannose), mixed into a solution, freeze-dried, and incubated at 55 °C for 3 days (72 h) at 65% relative humidity using saturated potassium iodide solution in a laboratory incubator. Native proteins were treated in a similar manner without mannose and regarded as the native, 9480

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Figure 2. Coomassie staining of SDS-PAGE patterns of native and glycated allergens. EW represents egg white, and EW-M represents mannosylated EW. PN represents peanut proteins, PN-M represents mannosylated PN, and PN-G represents glucosylated PN. Whey-M represents mannosylated whey. MW in all panels represents molecular weight markers (kDa). IgA was followed as described earlier11 except for the coating antigens replaced with EW, PN, and whey (10 μg/100 μL/well of antigens). Quantification of Specific Cytokines in Splenocytes. Post oral challenge, mice from each group were euthanized, and individual spleens were aseptically removed and minced. After erythrocyte lysis, spleen cells were resuspended in complete medium (RPMI 1640 containing 10% fetal bovine serum, 2 mM L-glutamine, 25 mM Hepes, 100 IU/mL penicillin, 100 mg/mL streptomycin). Spleen lymphocytes from individual mice were used in further analysis of cytokine measurements. Splenocyte suspensions at a concentration of 2.5 × 106 cells/well were daily stimulated with 50 μg/well of EW, PN, and whey antigens, respectively, for 72 h, whereas splenocytes from the mice treated with CT were added with 50 μL of saline as controls (unstimulated). The plates were covered and incubated at 37 °C in a 5% CO2 incubator with 95% humidity, and after 72 h, the aliquots were stored at −80 °C until further use. Concentrations of cytokines IFN-γ, IL-4, IL-10, IL-12, and TGF-β were determined using commercially available ELISA kits according to the manufacturer’s instructions as described earlier.11 Flow Cytometry. Flow cytometry was done using whole blood collected postchallenge from individual mice and pooled in duplicates (n = 4/group). Staining protocols were carried out as recommended by the manufacturer (BD Bioscience, San Jose, CA, USA). Samples were stained with 100 μL of FITC-conjugated anti-mouse CD25 antibody (558642; BD Bioscience) and phycoerythrin (PE)conjugated anti-mouse Foxp3 (clone FJKS-16; eBioscience) measured via a fluorescence activated cell sorter (FACScan Flow cytometer; BD Bioscience). After forward-scatter versus side-scatter gating, fluoroscence channels were analyzed and thresholds were set on isotype controls. Analysis was done using FCS Express 4 Plus software, and results were presented as percentage of double-stained CD25+ Foxp3+ cells. Statistical Analyses. All data were analyzed using GraphPad Prism software version 5.0 (GraphPad Software, San Diego, CA, USA). All data were expressed as means ± SEM and subjected to ANOVA analyses followed by post hoc multiple comparison using Tukey’s test. Comparison of all the end point differences with a level of p < 0.05 was considered significant.

List Biologicals, Campbell, CA, USA) for 4 weeks (twice a week) as presented in Figure 1. Mice were further treated with mannosylated and glucosylated antigens (1 mg) for 4 weeks (three times/week). At week 11 mice were orally challenged with 20 mg of each antigen and monitored for clinical signs of allergy as described earlier.11 Clinical scores were assigned in a blind fashion by three experienced observers. Total scores for each animal were obtained by adding scores for each individual sign. Blood was collected for measurement of histamine, antibody activity, and flow cytometry, and all samples were centrifuged for 20 min at 2000g and kept at 4 °C to obtain sera. All samples were stored at 20 °C until further analysis. Spleens were collected for measuring cytokines. Measurement of Serum Histamine and Mouse Mast Cell Protease. Concentration of serum histamine post oral challenge was determined using an enzyme immunoassay kit (Labor Diagnostika Nord, Nordhon, Germany) according to the manufacturer’s instructions. The mouse mast cell protease enzyme (MMCP) concentration was determined by ELISA as recommended by the manufacturer (eBioscience). Measurement of Allergen-Specific IgG, IgE, IgG1, and IgG2a. Serum allergen-specific IgE, IgG1, and IgG2a were detected by an indirect sandwich-type enzyme-linked immunosorbent assay (ELISA) using standard protocols as described earlier.11,15−17 In brief, 96-well microtiter plates (Costar, Corning Inc., NY, USA) were coated with 100 μL of EW, PN, and whey antigens (50 μg/mL in 50 mM carbonate buffer; NaHCO3/Na2CO3, pH 9.6) for 24 h at 4 °C. Rabbit anti-mouse IgG conjugated to alkaline phosphatase was used for the detection of IgG, and rat monoclonal anti-mouse IgE was used for IgE followed by streptavidin−HRP conjugate. For IgG1 and IgG2 rat monoclonal anti-mouse IgG1 and IgG2a were used followed by streptavidin−HRP conjugate. In brief, plates were washed and blocked with 2% bovine serum albumin/phosphate-buffered saline (PBS) at 37 °C for 1 h. Serum samples (diluted 1:10 for IgE measurement, 1:100 for IgG, IgG1, or IgG2a) in PBS containing 0.05% Tween 20 (PBS/ Tween) were added and incubated for 2 h at room temperature in duplicates. Plates were again washed, and the bound antibodies were detected by adding respective detection antibodies at room temperature for 1 h. Specific binding activity was detected by the addition of 100 μL/well of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma). The reaction was terminated with 2 N H2SO4 after 30 min, and absorbance at 415 nm was measured by a microtiter reader (Bio-Rad 550, Hercules, CA, USA). The data were expressed as percentage of positive control in optical density units. Percentage of positive control activity = (mean test OD/mean positive control serum OD) × 100. Determination of Specific IgA in Mouse Fecal Pellets. Antigen-specific IgA was measured in mouse fecal pellets collected freshly on a weekly basis at weeks 9, 10, and 11 and was processed as described earlier.11 Indirect ELISA procedure for measuring specific



RESULTS SDS-PAGE. Modifications of proteins due to the Maillard reaction in the presence or absence of CHO were observed as evidenced by the electrophoretic pattern. Figure 2 presents the SDS-PAGE pattern of the common allergens (EW, PN, and whey) before Maillard reaction (native, nonmodified protein) and after heating with allergen in the presence of mannose. All proteins heated in the presence of CHO appeared to be more

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IgG2 did not differ between groups (p > 0.05). Also, specific IgA as observed with glucosylated and mannosylated PN groups was less as compared to the native PN (p < 0.05) group. Specific IgA did not differ for the EW and whey groups (p > 0.05). Cytokine Concentration. Concentrations of type-1 cytokines IL-12 and IFN-γ, type-2 cytokine IL-4, regulatory cytokines IL-10 and TGF-β, and pro-inflammatory cytokine IL17A were measured. There was a significant decrease (Figure 5; p < 0.05) in IL-4 and a significant increase (Figure 5; p < 0.05) with IL-10 cytokines measured with the mannosylated EW group. Other groups did not differ significantly for the measured cytokines (p > 0.05). Proportion of T-Regulatory Cells. The proportion of blood T-regs was measured, and there was a significant increase in the percentage of CD25+ Foxp3+ cells as measured in the mannosylated EW group (Figure 6; p < 0.05). All other groups treated otherwise did not differ significantly (p > 0.05). A representative image for each treatment group is shown in Figure 6.

stable and were detected with a molecular weight slightly higher than that of the native protein, indicating occurrence of glycosylation. Moreover, slight aggregate formation was observed with PN and whey proteins, which was manifested by its presence at the boundary of the resolving and stacking gel. Nonglycated fractions were not visible, confirming negligible amounts of intact proteins in the glycated fractions. The binding ratios of the glycated fractions are given in Table 1. Table 1. Binding Ratio of the Conjugates glycated protein

CHO concn/protein concn ratio (w/w)

EW-M PN-M PN-G whey-M

3.85 6.67 6.67 8.00

Attenuation of Allergic Phenotype. Both clinical signs and clinical scores were less severe and frequent in the mannosylated EW group (Figure 3A; p < 0.05). Histamine concentration was significantly reduced in the mannosylated EW group (Figure 3B; p < 0.05), and MMCP concentration (Figure 3C; p < 0.05) was significantly reduced in both mannosylated EW and PN groups. All other groups did not differ significantly (p > 0.05). Immunoglobulin Isotype-Related Antibody Activity. Specific IgG, IgG1, and IgE isotypes were significantly different in the mannosylated EW group (Figure 4; p < 0.05). Specific



DISCUSSION

Structural changes of proteins that evolve due to conjugation of proteins with reducing sugars through the Maillard reaction are an effective way of improving the functionality of the protein, and this also seems to be a promising method for masking food protein allergenicity. We have for the first time, to the best of our knowledge, established that mannosylated EW plays a

Figure 3. (A) Clinical scores for individual mice. Total clinical scores for each mouse post oral challenge on week 11 were calculated. Average scores were assigned by three independent observers in a blind fashion. Treatment groups differed significantly (p ≤ 0.05; GraphPad Instat) in frequency of mice expressing allergic signs. (B) Serum histamine concentration. Data for serum histamine concentration are presented as mean ± standard deviation (n = 4 pooled sera). (C) Mouse mast cell protease (MMCP) concentration. Data for MMCP concentration are presented as mean ± SD. Different letters indicate statistically significant differences (p < 0.05) between groups of mice. 9482

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Figure 4. Antigen-specific IgG, IgE, IgG1, IgG2, and IgA-related antibody activity. Antigen-specific serum antibody activity was measured by ELISA in triplicates for each sample. Rabbit anti-mouse IgG conjugated to alkaline phosphatase was used for the detection of IgG, and rat monoclonal antimouse IgE was used for IgE followed by streptavidin−HRP conjugate. For IgG1 and IgG2 rat monoclonal anti-mouse IgG1 and IgG2a were used followed by streptavidin−HRP conjugate. IgA was detected from pooled fecal samples collected during weeks 9−11 using biotinylated rat monoclonal anti-mouse IgA antibody followed by avidin−HRP conjugate. Data are presented as percentage of positive control (sera from mice that had high Ovm-specific IgG) activity, calculated as [optical density (OD) of test serum]/(OD of positive control serum − OD of negative control) × 100%. Significance was taken at p ≤ 0.05. Different letters indicate statistically significant differences.

critical role in attenuating the development of allergic sensitization. Mice were significantly protected against most of the aspects of allergic inflammatory response for the biomarkers tested. There was a significant difference in the IR phenotype measured with the mannosylated EW group, such that the mice had reduced clinical scores, histamine, and MMCP. Mice also had less specific IgG and IgE as compared to the EW group. Cytokine profiles revealed that the mannosylated EW group had less IL-4 and more IL-10. The proportion of T-regs differed significantly from those of the native EW

group. It may be postulated that structural changes to mannosylated EW may have affected the stability and uptake of the protein, thereby altering its digestability and absorption, and this may have resulted in reduction of its allergenic potential. Mannosylated PN and whey did not attenuate allergic signs, indicating that these proteins do not functionally interact to augment inhibition of the effector phase of the allergic inflammatory response and did not appear to be important determinants of modulating the outcomes of allergic signs. It 9483

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Figure 5. Cytokine concentration. Spleens were collected from each mouse and pooled within groups (n = 4), and splenocytes were isolated and cultured at 2.5 × 106 cells/mL concentration of cells in triplicate wells as unstimulated (control) or stimulated with 50 μg/well each antigen for 72 h. The cytokine concentration of cell culture supernatants for IL-4, IFN-γ, IL-12, IL-10, TGF-β, and IL-17 was determined by ELISA. Different letters indicate significant difference between groups for each cytokine. Significance was taken at p ≤ 0.05.

the mannosylated PN group, so this hypothesis requires further studies. Effects of heat treatment on whey protein isolate (WPI) were reported earlier by Bu et al.,23 and they showed that the antigenicity of WPI increased with increasing temperature from 50 to 90 °C. However, antigenicity remarkably decreased above 90 °C. This was similar to the observation in this study that whey protein incubated at 55 °C for 3 days did not decrease allergenicity in vivo. It may be possible that Maillard reaction may have led to the exposure of allergenic whey protein epitopes of the native molecule due to the unfolding of conformational structure. New epitopes may have emerged as a result of the conjugation process and led to the exposure of the hydrophobic region. On the other hand, effects of Maillard reaction of WPI with glucose were investigated earlier using response surface methodology, and the results demonstrated that the conjugation of WPI with glucose effectively reduced

was earlier demonstrated that extracts from roasted PN were shown to bind higher IgE levels than extracts from raw PN,20 and other studies have supported a role of advanced glycated products in enhancing IgE binding.21 This is in good agreement with the study conducted here that both glucosylated and mannosylated PN did not alleviate clinical signs of allergy in Balb/c mice. Most of the biomarkers tested for allergy did not differ significantly as compared to the native PN antigen group, except for reduced MMCP concentration as observed in the mannosylated PN group. MMCPs under normal/unprovoked conditions are sequestered within the MC granule, and degranulation of MC leads to the massive release of fully active MC proteases.22 An inhibitory allergic effect may result from reduced concentration of MMCP; however, in this case, although protein mediator releasing capacity was low with MMCP, it is unclear what contibuted to overall allergic signs of 9484

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Figure 6. Flow cytometry. Percentage of CD25+ Foxp3+ cells was determined by FACS from whole blood of mice collected post oral challenge. A representative image of CD25+ Foxp3+ cells for each group and respective percentage of cells for each quadarant are shown.

the antigenicity.24 It was also shown earlier that that the antigenicity of β-lactoglobulin and bovine serum albumin, major allergens in WPI, were significantly reduced by glycation with maltopentaose (MP) or glycation and phosphorylation, respectively.25,26 Several studies have earlier demonstrated that antigen mannosylation or mannosylated Ag delivery systems, such as mannosylated liposomes, could enhance not only MHC class II- but also MHC class I-restricted Ag presentation and T cell stimulation by targeting mannose receptors on Ag-presenting cells.27,28 It was also shown that oligomannose-coated liposome ameliorates allergic signs in OVA-sensitized allergic mice.29 In this study, the Maillard reaction induced changes in mannosylated EW allergic functionality. We speculate that the loss of secondary structure may have led to the destruction of conformational epitopes and thus to a decreased IgE binding capacity, and this may have had an effect on EW allergenicity. Also, mannosylation may have reduced the digestibility and absorption of the EW allergen in mouse. It was earlier reported

that glycation of EW with MP reduced antiovomucoid and antiOVA antibody response, and glycation and phosphorylation of EW by dry-heating overall improved the functional properties of EW protein.30 We had demonstrated earlier via mannosylation of OVA that a change in secondary structure and processing via dendridic cell uptake and T cell activation led to alleviated allergic signs in mice.11 However, circular dichroism for measuring secondary structure of EW, PN, and whey was not performed here due to the complex nature of the proteins. It was earlier shown that the transfer of CD25+ T regs suppresses Th2-dependent reactions to allergens in vivo by an IL-10-mediated pathway and is thus able to reduce airway hyper-reactivity in a mouse model.31 It is also known that the skew from type-2 allergic response to T reg predominance and influence of T reg-derived IL-10 response are known to strongly suppress allergen-specific IgE production.32 Work done here confirms this and supports the concept that T regderived IL-10 may have used a similar molecular pathway to suppress allergen-specific IgG, IgG1, and IgE with mannosy9485

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product, shows enhanced T-cell immunogenicity. J. Biol. Chem. 2014, 289, 7919−7928. (11) Rupa, P.; Nakamura, S.; Katayama, S.; Mine, Y. Effects of ovalbumin glycoconjugates on alleviation of orally induced egg allergy in mice via dendritic-cell maturation and T-cell activation. Mol. Nutr. Food Res. 2013, 58, 405−417. (12) Gazi, U.; Martinez-Pomares, L. Influence of the mannose receptor in host immune responses. Immunobiology 2009, 214, 554− 561. (13) Royer, P. J.; Emara, M.; Yang, C.; Al-Ghouleh, A.; Tighe, P.; Jones, N.; Sewell, H. F.; Shakib, F.; Martinez-Pomares, L.; Ghaemmaghami, A. M. The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergeninduced T cell polarization through modulation of IDO activity. J. Immunol. 2010, 185, 1522−1531. (14) Al-Ghouleh, A.; Johal, R.; Sharquie, I. K.; Emara, M.; Harrington, H.; Shakib, F.; Ghaemmaghami, A. M. The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent. PLoS One 2012, 7, No. e33929. (15) Jiménez-Saiz, R.; Rupa, P.; Mine, Y. Immunomodulatory effects of heated ovomucoid-depleted egg white in a BALB/c mouse model of egg allergy. J. Agric. Food Chem. 2011, 59, 13195−13202. (16) Rupa, P.; Hamilton, K.; Cirinna, M.; Wilkie, B. N. Porcine IgE in the context of experimental food allergy: purification and isotypespecific antibodies. Vet. Immunol. Immunopathol. 2008, 125, 303−314. (17) Li, E. W. Y.; Mine, Y. Comparison of chromatographic profile of glycomacropeptide from cheese whey isolated using different methods. J. Dairy. Sci. 2004, 87, 174−177. (18) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric method for determination of sugars and related substances. J. Anal. Chem. 1956, 28, 350−356. (19) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76−85. (20) Maleki, S. J.; Chung, S. Y.; Champagne, E. T.; Raufman, J. P. The effects of roasting on the allergenic properties of peanut proteins. J. Allergy Clin. Immunol. 2000, 106, 763−768. (21) Nesbit, J. B.; Hurlburt, B. K.; Schein, C. H.; Cheng, H. P.; Wei, H.; Maleki, S. J. Ara h 1 structure is retained after roasting and is important for enhanced binding to IgE. Mol. Nutr. Food Res. 2012, 56, 1739−1747. (22) Pejler, G.; Rönnberg, E.; Waern, I.; Wernersson, S. Mast cell proteases: multifaceted regulators of inflammatory disease. Blood 2010, 115, 4981−4990. (23) Bu, G. H.; Luo, Y. K.; Zheng, Z.; Zheng, H. Effect of heat treatment on the antigenicity of bovine α-lactalbumin and βlactoglobulin in whey protein isolate. Food Agric. Immunol. 2009, 20, 195−206. (24) Bu, G. H.; Lu, J.; Zheng, Z.; Luo, Y. K. Influence of Maillard reaction conditions on the antigenicity of bovine α-lactalbumin using response surface methodology. J. Sci. Food Agric 2009, 89, 2428−2434. (25) Enomoto, H.; Li, C. P.; Morizane, K.; Ibrahim, H. R.; Sugimoto, Y.; Ohki, S.; Ohtomo, H.; Aoki, T. Glycation and phosphorylation of β-lactoglobulin by dry-heating: effect on protein structure and some properties. J. Agric. Food Chem. 2007, 55, 2392−2398. (26) Enomoto, H.; Li, C. P.; Morizane, K.; Ibrahim, H. R.; Sugimoto, Y.; Ohki, S.; Ohtomo, H.; Aoki, T. Improvement of functional properties of bovine serum albumin through phosphorylation by dryheating in the presence of pyrophosphate. J. Food Sci. 2008, 73, 84−91. (27) Sheng, K. C.; Kalkanidis, M.; Pouniotis, D. S.; Esparon, S.; Tang, C. K.; Apostolopoulos, V.; Pietersz, G. A. Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo. Eur. J. Immunol. 2008, 38, 424−436. (28) Zhou, X.; Liu, B.; Yu, X.; Zha, X.; Zhang, X.; Wang, X.; Jin, Y.; Wu, Y.; Chen, Y.; Shan, Y.; Chen, Y.; Liu, J.; Kong, W.; Shen, J. Controlled release of PEI/ DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune

lated EW. Also, Aoki et al. have earlier confirmed that attachments of mannose to Cry j 1 allergen increased the colocalization of antigen with immune cells, such as dendritic cells, which have a mannose receptor, and it is expected that Cry j 1−mannnose conjugate is presented to antigen-presenting cells, such as dendritic cells, by mediation of a mannose receptor, leading to sequent antigen presenting to regulatory T cells.33 This may be one of the possible mechanisms occurring here for increased frequency of T reg cells in the EW-M group. Overall, this study reveals that mannosylated EW effectively suppressed orally induced allergic IR in mice. Several pieces of mechanistic evidence as derived from this study suggest that mannosylated EW modifies numerous surrogate clinical markers of allergy and validates a practical approach to cure egg allergy. Nevertheless, it also concludes that this approach does not seem to apply to any given allergen such as PN or whey. These results could have important implications for therapeutic use and potentially be targeted as a valid strategy for the effective treatment of egg allergy.



AUTHOR INFORMATION

Corresponding Author

*(Y.M.) Phone: (519) 824-4120, ext. 52901. Fax: (519) 8246631. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



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