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SENSITIZING AND ELICITING CAPACITY OF EGG WHITE PROTEINS IN BALB/C MICE AS AFFECTED BY PROCESSING Alba Pablos-Tanarro, Daniel Lozano-Ojalvo, Mónica Martínez-Blanco, Rosina López-Fandiño, and Elena Molina J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017
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Journal of Agricultural and Food Chemistry
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SENSITIZING AND ELICITING CAPACITY OF EGG WHITE PROTEINS IN BALB/C
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MICE AS AFFECTED BY PROCESSING
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ALBA PABLOS-TANARRO, DANIEL LOZANO-OJALVO, MÓNICA MARTÍNEZ-
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BLANCO, ROSINA LÓPEZ-FANDIÑO, ELENA MOLINA
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Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Madrid, Spain
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[email protected];
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[email protected] [email protected];
[email protected];
[email protected];
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Correspondence: Elena Molina
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CIAL, Nicolás Cabrera 8, 28049 Madrid, Spain
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E-mail:
[email protected] 13
Phone: +3491 0017938
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FAX: + 34 91 0017905
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Abbreviated running title: Allergenicity of processed egg white proteins
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ABSTRACT
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This study assesses to what extent technological processes that lead to different degrees of
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denaturation of egg white proteins affect their allergenicity. We focused on heat (80ºC, 10 min)
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and high pressure (400 MPa and 37ºC, 10 min) treatments and used a BALB/c mouse model of
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food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the
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production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with
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the allergen. Extensive protein denaturation caused by heat treatment increased its ability to
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induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white
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stimulated the production of IgE over IgG1 antibodies directed, at least in part, towards new
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epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high
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pressure treatment increased the ability of egg white to stimulate Th2 responses and its
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allergenic potential.
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KEY WORDS: egg allergy; heat-treatment; high-pressure; BALB/c; sensitization; anaphylaxis;
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IgE; IgG1
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Introduction
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Food allergy is a major health problem in Western countries, affecting, approximately, 5%
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of adults and 8% of children and egg proteins are the second leading cause of allergy during
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infancy and early childhood.1 Although it is a complex scenario, it is broadly accepted that the
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prevalence and severity of food allergies are increasing alarmingly and the economic and social
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impact is growing. In addition, so far, there is no approved treatment for food allergy and
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sensitized individuals need to avoid ingestion of food to which they are allergic.2
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Thermal processing techniques, commonly used during food production, have the potential
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to impact food allergens by inducing structural alterations, such as unfolding and aggregation.
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The effects of temperature on different food allergens have been extensively investigated and
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there is a general agreement that it is very important to understand how heat treatments alter the
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structure of food proteins and the subsequent gastrointestinal digestibility, both of them
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influencing their allergenicity.3-5 Other processing techniques, such as high hydrostatic pressure
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can also lead to denaturation of proteins depending on the pressure level, temperature, and
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chemical conditions. Johnson et al.
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structure and aggregation state of a selection of purified food allergens after high pressure
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application, although, depending on the protein, pressurization may induce sufficient structural
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modifications to affect susceptibility to digestion and immunoreactive properties, lowing or
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enhancing the binding to IgE and the capacity to trigger mast cell activation and produce
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clinical reactions.4
6
did not observe substantial changes in the secondary
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Physicochemical changes caused by heat treatment on egg proteins are often associated
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with a decrease in their allergenicity.7 Heating of egg proteins increases the digestibility of
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ovalbumin (OVA), the most abundant egg allergen, and lowers the binding of egg allergens to
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IgE from patient sera;8-10 although the reactivity of IgE from egg allergic patients towards the
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native or heated forms varies depending not only on whether egg has been extensively of
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partially cooked, but also on their individual susceptibility.11 However, while oral exposure to
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cooked eggs is likely to be the most frequent source of immunization, the sensitizing potential
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of heated proteins administered by the oral route has not been studied in depth. Regarding high3 ACS Paragon Plus Environment
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pressure treatments, Hildebrandt et al.,12 reported that the IgE-binding of eggwhite allergens in
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meat products decreases with increasing pressure, from 400 to 700 MPa. It is also known that
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simultaneous enzymatic treatment and pressurization increases the susceptibility of OVA to
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hydrolysis by pepsin, trypsin and chymotrypsin.13 Nevertheless, the information available does
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not allow drawing a clear picture of the effect of mild denaturation treatments on the
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allergenicity of egg and, in fact, there are no in vivo data on the allergenic potential of
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pressurized egg proteins.
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The aim of this study was to investigate to what extent technological processes that lead to
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different degrees of denaturation of egg proteins affect their sensitization and elicitation ability
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in a mouse model. We have focused on moderate heat and high pressure treatments, applied,
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respectively, at 80ºC and 400 MPa for 10 min; which are usual operating conditions in food
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processing whose effects on the allergenic potential of egg white have not been previously
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addressed. Raw, heated and pressurized egg white (EW, HEW and PEW, respectively) were
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orally administered to BALB/c mice. Serum antibody levels were recorded and severity of
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anaphylaxis was evaluated following allergen challenge. Special attention was paid to the
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specificity of the generated antibodies and the systemic cytokine profiles.
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Materials and methods
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Proteins, chemicals and enzymes for in vitro digestion were purchased from Sigma–
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Aldrich (St. Louis, MO, USA) unless otherwise specified. Antibodies were from BD
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Biosciences (San Diego, CA, USA). Eggs came from organic-crop fed poultry and were
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purchased from a local supermarket.
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Egg white proteins
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Egg white (EW), separated from fresh eggs and commercial egg proteins -ovalbumin grade
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V (OVA), ovomucoid type III-O (OM) and lysozyme (LYS)- were used. Heated egg white
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(HEW) was obtained by heating at 80ºC for 10 min and pressurized egg white (PEW) by high
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hydrostatic pressure treatment (High Pressure Equipment, Stansted ISO-LAB system, Essex, 4 ACS Paragon Plus Environment
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UK) at 400 MPa for 10 min at 37ºC. Samples were freeze dried, analysed for protein content by
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the Kjeldahl method (85.12%, 84.70% and 84.23% in EW, HEW and PEW, respectively) and
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structurally characterized by SDS-PAGE and circular dichroism (CD). The lipopolysaccharide
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content (analysed by the Pierce® LAL Chromogenic Endotoxin Quantitation Kit, Thermo
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scientific, Waltham, MA, USA) was below of 1 UE/mg in all proteins and egg white samples
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except for OVA, which was purified by size exclusion chromatography.14
97 98
Structural analysis by SDS-PAGE and CD
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Samples for SDS-PAGE were dissolved at 2 mg of protein/ml in sample buffer that
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contained 50 mM Tris-HCl (pH 6.8), 10% v/v glycerol, 2% w/v SDS and 0.002% w/v
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bromophenol blue, in the absence or presence of 5% β-mercaptoethanol and heated for 10 min
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at 95ºC. Electrophoretic separations were carried out at 120 V on Precast Criterion XT 12% Bis-
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Tris gels (Bio-Rad, Hercules, CA, USA) using XT MES as running buffer (Bio-Rad). Precision
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Plus Protein Unstained Standard was used as molecular weight marker. Gels were stained with
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Coomasie Blue, and images were taken with a Molecular Imager Versadoc MP 5000 system and
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processed using Quantity One 1-D analysis software (all from Bio-Rad).
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CD was performed as previously described by Benedé et al.15 Far (200-250 nm) CD spectra
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of EW, HEW and PEW, dissolved at 0.2 mg of protein/ml in phosphate buffer 50 mM pH 7.0,
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were recorded at 20ºC using cells with 0.1 cm pathlengths in a Jasco J-810 spectropolarimeter
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(Jasco Corp., Tokyo, Japan). Spectra represent the average of three accumulations collected at
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20 nm/min, with a 2 s time constant, a 0.2 nm resolution and a sensitivity of 100 mdeg. Buffer
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blanks were subtracted from each CD spectrum, which were represented as mean specific
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ellipticity (degree·cm2·cg-1). CDNN secondary structure analysis software (Applied
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Photophysics Ltd, Surrey, UK) was used.
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In vitro gastroduodenal digestion
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In vitro digestions were carried out as previously described.16 Briefly, gastric digestions
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were performed at a protein concentration of 6.4 mg/ml in simulated gastric fluid (35 mM NaCl, 5 ACS Paragon Plus Environment
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pH 2.0) for 60 min at 37ºC, with 182 units/mg protein of porcine pepsin (EC 3.4.23.1, 4220
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U/mg protein). Aliquots were withdrawn at 20 and 60 min and reactions stopped by raising the
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pH up to 7.0-7.5 with NaHCO3. For intestinal digestions, the gastric digests were mixed with
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0.25 M Bis-Tris, pH 6.5, 1 M CaCl2 and a 0.125 M bile salt mixture, containing equimolar
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quantities of sodium taurocholate and glycodeoxycholic acid. Pancreatic bovine trypsin (EC
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232-650-8, type I) and α-chymotrypsin (EC 232-671-2; type I–S), and pancreatic porcine lipase
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(EC 232-619-9; type VI-S) were added to the mixture at enzyme: protein ratios of 34.5, 0.4 and
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24.8 units per mg of protein, respectively. Pancreatic porcine colipase (EC 259-490-1) was
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added at an enzyme:protein ratio of 1:895 (w:w). Duodenal digestions were carried out for 20
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and 60 min at 37ºC and stopped by adding trypsin-chymotrypsin inhibitor, at a concentration
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calculated to inhibit twice the amount of trypsin and chymotrypsin present in the digestion mix.
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The digests were immediately mixed with SDS-PAGE sample buffer and heated as indicated
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above.
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Animals
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Six-week-old female BALB/c mice (Charles River Laboratories, Saint Germain sur
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l´Abresale, France) were kept under specific-pathogen-free conditions and fed an animal
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protein-free diet (SAFE, Route de Saint Bris, France) and water ad libitum. All protocols
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involving animals were approved by the CSIC Bioethics Committee and the Comunidad de
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Madrid (Ref PROEX 089/15) and the European legislation (Directive 2010/63/UE).
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Sensitization and challenge protocols
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Mice (distributed in 8 groups of 5 animals) were orally administered the amount of EW (3
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groups), HEW (2 groups) or PEW (2 groups) equivalent to 5 mg of protein plus 10 µg of CT
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(List Biologicals, Campbell, CA, USA), or just PBS (naïve group). Sensitization was performed
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during 3 consecutive days on the first week and once a week during the following 6 weeks. On
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week 8, mice were orally (50 mg of protein) and intraperitoneally (i.p., 100 µg of protein)
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challenged, 40 min apart. EW-sensitized mice were challenged with EW, HEW and PEW; 6 ACS Paragon Plus Environment
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HEW-sensitized mice, with EW and HEW; and PEW-sensitized mice with EW and PEW. Naïve
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mice were challenged just with PBS. Anaphylactic signs and body temperature drops were
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evaluated 30 min after the oral and i.p. challenges.17 All mice were euthanized at the end of the
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challenge protocol by CO2 inhalation.
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At different experimental points, blood samples were collected by cheek puncture and
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centrifuged at 2000 x g for 15 min. Serum levels of mouse mast cell protease-1 (MCP-1) were
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quantified post-mortem with a commercial ELISA kit (eBioscience, San Diego, USA), as
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outlined by the manufacturer.
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Passive cutaneous anaphylaxis (PCA)
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Following Li et al.,18 20 µl of pooled samples of sera collected post-mortem from five
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HEW-sensitized mice, either unheated or heat-inactivated (56ºC for 3 h), were intradermically
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inoculated in the right ear pinna of BALB/c naïve mice (2 mice per treatment). Pooled serum
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from naïve mice was inoculated in the left ear pinna. Twenty-four hours later, mice were
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intravenously injected with 200 µg on a protein basis of HEW in 100 µl of 0.5% Evans blue dye
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(Sigma-Aldrich). Sixty minutes apart, mice were euthanized by CO2 inhalation and ears were
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individually collected, weighed and incubated overnight at 55ºC in N,N-dimethylformamide
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(Sigma-Aldrich). The absorbance of the supernatant was measured at 655 nm, corrected with
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the absorbance of the supernatant from the ear injected with pooled naïve sera, and compared
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with a standard curve of serial dilutions of Evans blue, in order to quantify dye extravasation per
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g of mouse ear.
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Indirect ELISA for the detection of specific IgE and IgG1
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Indirect ELISA was performed by coating 96-well plates with 5 µg/ml (for IgE) or 2 µg/ml
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(for IgG1) of protein (OVA, OM, LYS, EW, HEW and PEW).17 To build reference curves, plates
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were coated with rat anti-mouse IgE and IgG1 (BD Biosciences). Blocked plates were incubated
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overnight at 4ºC with serum samples (1/25 diluted for IgE and 1/1000 or 1/5000 diluted for
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IgG1). Serial dilutions of mouse IgE and IgG1 were used for the reference curves (BD 7 ACS Paragon Plus Environment
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Biosciences). Subsequently, plates were incubated with biotin rat anti-mouse IgE and IgG1 and
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streptavidin-HRP (BD Biosciences). Colorimetric reactions were read at 405 nm in a plate
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reader
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ethylbenzothiazoline-6-sulfonic acid) as substrate (Roche, Mannheim, Germany).
(Multiskan
FC,
Thermo
Scientific)
after
addition
of
2,2′-Azino-bis(3-
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Antibody-capture ELISA for the study of the IgE binding capacity
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EW, HEW and PEW were labelled with EZ-link sulpho-NHS-LC biotin (Pierce, Rockford,
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IL, USA) using a 5:1 molecular ratio of biotin:protein. Pooled sera from 5 mice sensitized to
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EW, HEW or PEW (1/100 diluted) were added to plates coated with rat anti-mouse IgE (2
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µg/ml, clone MCA 419, BioRad) and incubated overnight at 4ºC. For direct ELISA, 50 µl of
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biotin-labelled antigen (500 ng/ml) were added and incubated for 3 h at room temperature. In
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the case of the competition assays, mixtures of 25 µl of biotinylated antigen and 25 µl of diluted
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non-labelled antigen (from 1 ng/ml to 0.5 mg/ml) were added to the plates. After incubation
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with streptavidin-HRP (BD Biosciences), 3,3′,5,5′-tetramethylbenzidine was added as substrate
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(eBioscience). The colorimetric reaction was stopped with H2SO4 and read at 450 nm in a plate
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reader (Multiskan FC, Thermo Scientific). Results were expressed as B/B0, where B0 and B
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represent, respectively, the amount of labelled EW, HEW or PEW bound to the immobilized
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IgE antibodies in the absence or presence of a known concentration of non-labelled EW,
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HEW or PEW.
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Cytokines released by spleen cells
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Spleen from individual mice were collected and processed as previously described.19
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Isolated splenocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine
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serum, 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin (all from Biowest
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SAS, Nuaillé, France) at a cellular density of 4x106 cells/ml in 48-well plates (non-stimulated
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control) and incubated with 200 µg/ml (on a protein basis) of OVA, OM, LYS, EW, HEW or
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PEW. Supernatants were collected after 72 h of culture in 5% CO2 at 37 °C and stored at -80 °C
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until ELISA analyses (eBioscience) to quantify cytokine production(IFN-γ, IL-4, IL-5 and IL-
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10).
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Statistical analyses
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Experimental results are expressed as means ± standard error of the mean (SEM).
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Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by
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Tukey’s test for comparing all groups. Clinical scores are expressed as medians and, in this
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case, significant differences were determined using the unpaired non-parametric Mann-Whitney
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test. T-test was used to test differences between unheated and heat-inactivated sera values in the
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PCA test. The statistical software package GraphPad Prism version 6.0 (GraphPad Software, Inc
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La Jolla, CA, USA) was used for the analyses. P < 0.05 was considered statistically significant.
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Results
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Structural changes and digestibility of egg white proteins as a result of heat and high pressure
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treatments
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SDS-PAGE analysis under non reducing conditions revealed similar patterns for EW and
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PEW, although HEW showed a completely different profile characterized by the presence of
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high molecular mass aggregates and the absence of bands corresponding to the main EW
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proteins (Fig. 1a). At least part of these aggregates were reduced following the addition of β-
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mercaptoethanol, with the appearance of bands corresponding to ovotransferrin (OVT), OVA,
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OM and LYS, which indicates that they were stabilized by disulphide bonds induced by
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exchanges between free sulfhydryl and disulphide groups of these proteins, while other
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aggregates, possibly stabilized by non-reducible covalent bonds, remained undissociated (Fig.
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1a).
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To unveil further structural differences after processing, far-UV CD spectra were collected
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and compared (Fig. 2). The spectra of HEW confirmed secondary structure changes as a result
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of heat treatment. CD also showed that PEW was structurally similar to EW with respect to the
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secondary structure content.
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Following simulated gastrointestinal digestion, there was an incomplete degradation of EW
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and PEW proteins (Fig. 1b). In particular, OVA partially resisted successive gastric and
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duodenal in vitro hydrolyses. However, HEW was more susceptible to proteolysis and neither
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aggregates nor individual native proteins were detected in the SDS-PAGE gels at the end of
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digestion process.
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Sensitizing and eliciting potential of processed egg white proteins
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Serum concentrations of IgE and IgG1 specific to EW and to the individual proteins, OVA,
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OM and LYS, were determined along the sensitization period by indirect ELISA
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(Supplementary Fig. 1). Table 1, which compares the antibody levels at the 8th week just before
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challenge, indicates that HEW-sensitized mice presented a higher amount of EW-specific IgE
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than mice sensitized to EW. In particular, the OVA-specific IgE response was greater in HEW-
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sensitized than in EW-sensitized mice, although the LYS-specific IgE response was lower. In
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addition, HEW-specific IgE was also detected in serum from HEW-sensitized mice at a higher
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concentration than that of EW-specific IgE. Although the difference did not reach statistical
244
significance, this suggests the formation of IgE antibodies directed towards different antigenic
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determinants exposed by heat treatment. There were no significant differences in the levels of
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EW-, OVA- and OM-specific IgE between EW- and PEW-sensitized mice, although LYS-
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specific IgE was the highest in mice sensitized to PEW. Sensitization to PEW generated
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comparable amounts of EW- and PEW-specific IgE (Table 1).
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Sensitization to HEW did not lead to a significant EW-specific IgG1 response as compared
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with naïve mice. Furthermore, the concentration of HEW-specific IgG1 in the serum of HEW-
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sensitized mice was also low (Table 1). However, oral administration of EW and PEW plus CT
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significantly increased the levels of EW-specific IgG1, with PEW-sensitized animals showing
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equivalent amounts of EW- and PEW-specific IgG1. We detected similar concentrations of
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OVA-, OM- and LYS-specific IgG1 within each mouse group, and no differences between both
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The ability to trigger anaphylactic reactions of HEW and PEW, as compared to EW, was
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assessed in mice sensitized to EW (Fig. 3). Oral challenge with HEW did not cause a significant
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drop in temperature as compared with naïve mice administered PBS, nor did challenge with
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EW, while PEW significantly decreased body temperature in mice (Fig. 3a). Systemic
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anaphylactic symptoms were evident in all cases, but there were no significant differences
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among EW-sensitized mice challenged with the EW preparations submitted to processing under
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different conditions (Fig. 3b). Similar results were obtained after subsequent i.p. challenges,
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although, in this case, HEW produced the least severe clinical signs. Challenge with HEW also
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led to the lowest release of MCP-1, indicative of mast cell degranulation (Fig. 3c). Overall,
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these results imply that HEW and PEW induced, respectively, weaker and stronger anaphylactic
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responses than EW in EW-sensitized mice.
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The lower capacity of HEW, as compared with EW, to elicit systemic reactions was also
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evident in the sign score of mice sensitized to HEW and challenged either orally or i.p. with
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both preparations. In fact, mice sensitized to HEW did not suffer temperature changes nor
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anaphylactic sings following challenge with HEW (Fig. 3a and b). However, both PEW and EW
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caused similar temperature drops, allergic symptoms and MCP-1 release in PEW-sensitized
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mice. It is noteworthy that significant temperature drops and MCP-1 levels were only detected
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in mice sensitized to PEW (and challenged either with EW or PEW), as well as in mice
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sensitized to EW and challenged with PEW, which suggests that, besides a superior ability to
275
trigger allergic reactions, PEW held an enhanced sensitization potential.
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3.3. Specificity of the generated antibodies
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As mentioned, serum levels of MCP-1 were significantly increased in mice sensitized to
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EW and PEW (and challenged, respectively, with PEW and either EW or PEW), but not in mice
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sensitized to HEW (Fig. 3c), despite the later exhibited the highest specific IgE levels (Table 1).
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This observation prompted us to investigate the ability of IgE generated in HEW-sensitized
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mice to develop allergic responses. To this aim, we conducted PCA assays with pooled serum
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heated at 56°C to inactivate mouse IgE antibodies. 18 Passive immunization of naïve mice with 11 ACS Paragon Plus Environment
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heated serum from HEW-sensitized mice led to a significantly reduced extravasation of Evan’s
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blue dye to the surrounding tissues upon intravenous challenge with HEW, as compared with
286
the immunization using non-heated serum, denoting that IgE was biologically functional and
287
able to mediate local anaphylaxis reactions (Supplementary Fig. 2).
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To go deeper into this point, we assessed the binding capacity of IgE present in sera from
289
EW-, HEW- and PEW-sensitized mice to EW subjected to different processing methods using a
290
reverse ELISA based on the capture of mouse IgE antibodies. Initially, we compared the
291
binding capacity of the immobilized IgE molecules towards the antigen used for sensitization in
292
each case by a direct test, which confirmed a higher response in HEW-sensitized mice (Fig. 4a).
293
Competitive ELISA tests, showed that IgE from sera of mice sensitized to HEW bound HEW
294
with a much higher strength than EW and PEW, underlining that sensitization to HEW
295
generated IgE antibodies specific to different or new epitopes (Fig. 4 c). As mentioned above,
296
the observation that, despite allergy in HEW-sensitized mice was mainly IgE-mediated and
297
HEW-specific IgE displayed higher affinity for HEW than for EW (Supplementary Fig. 2 and
298
Fig. 4c), HEW triggered less severe allergic responses than EW in these animals following both
299
oral and systemic challenges (Fig. 3) shows that heating impaired its ability for mast cell
300
crosslinking and activation, given that challenge with EW was able to trigger anaphylactic
301
reactions in HEW-sensitized mice
302
On the other hand, as illustrated in Fig. 4b and d, both EW and PEW inhibited with similar
303
affinity the binding of IgE from sera of EW or PEW-sensitized mice to labelled EW and PEW
304
respectively, whereas HEW was a much weaker inhibitor, particularly of the binding of specific
305
IgE to biotinylated EW. This reveals that heat treatment destroyed IgE-binding epitopes present
306
in EW and that these were maintained when EW was submitted to less severe denaturation
307
conditions, such as 400 MPa applied for 10 min. Additionally, it denotes that sensitization to
308
both EW and PEW induced the production of IgE antibodies with similar specificities.
309 310
3.4. T cell responses in spleen cell cultures
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Splenocytes from EW-, HEW- and PEW-sensitized mice were stimulated with the 3
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protein preparations, as well as with the main egg allergens, OVA, OM and LYS (Fig. 5).
313
Cytokines produced in cultures from naïve mice were undetectable (results not shown). In
314
general terms, spleen cells from sensitized mice within each group released equivalent amounts
315
of IFN-γ, IL-4, IL-5 and IL-10 in response to either the individual proteins or the raw and
316
processed EW forms, with the latter stimulating the highest production. However, splenocytes
317
from HEW-sensitized mice were more prone to Th1 responses, as judged by the release of IFN-
318
γ to the culture media, and those from PEW-sensitized mice released more Th2 (IL-4 and IL-5)
319
cytokines and IL-10, following incubation with the stimuli.
320 321
4. Discussion
322
In this study, the ability of EW subjected to denaturation by means of heat treatment (80ºC,
323
10 min) and high hydrostatic pressure (400 MPa, 10 min) to sensitize and trigger allergic
324
responses was compared in BALB/c mice. Thermal treatment affected egg protein secondary
325
structure and caused polymerization, as determined by CD and SDS-PAGE, in agreement with
326
Mine et al.,20 who described a marked increase in β-sheet and a concomitant decrease in α-helix
327
structure, exposure of hydrophobic residues and aggregation by disulphide bridges with
328
increasing temperature from 60 to 90ºC. On the other hand, and also in accordance with our
329
results, few changes in the secondary structure of OVA and LYS have been reported between
330
the native proteins and those pressurized up to 600 MPa.21-22 High pressure treatments can result
331
in a pressure-dependent exposure of buried sulfhydryl groups leading to EW protein
332
aggregation, although at 40ºC this only occurs above 600 MPa.23 Therefore, the heat and high
333
pressure processing conditions applied led, respectively, to extensive and limited denaturation
334
of EW proteins. Assessment of resistance to simulated gastrointestinal digestion, which is
335
another indicator of structural stability, revealed that, as expected, HEW was far more prone to
336
hydrolysis than EW, which partially withstood digestion, as previously reported for heated and
337
native OVA,9,24-25 whereas EW and PEW exhibited similar susceptibility to proteolysis.
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338
Sensitization to HEW induced IgE antibodies specific to native and denatured EW proteins.
339
In fact, the serum concentration of EW-specific IgE, as determined by indirect ELISA, was
340
significantly higher in HEW-sensitized mice than in EW-sensitized mice, and that of HEW-
341
specific IgE was even greater. Since indirect ELISA does not avoid interactions of plate bound
342
antigens with specific IgG1, which may impair the binding and detection of IgE antibodies, a
343
selective IgE capture ELISA was conducted.26 Even though biotin labelling may vary depending
344
on protein structure, hindering comparison, this method also showed that severe denaturation of
345
EW proteins caused by heat treatment stimulated the production of IgE antibodies which were,
346
at least in part, different in their specificity from those produced upon sensitization with native
347
allergens. In addition, PCA assays revealed that such IgE antibodies were biologically
348
functional in the development of allergic reactions. However, even if HEW was able to generate
349
the most vigorous IgE response, its eliciting capacity, both in EW- and HEW-sensitized mice
350
was very low.
351
It is well documented that heat treatment of egg proteins decreases their allergenic
352
potential. Approximately 70% of egg allergic children tolerate extensively heated eggs and, in
353
these cases, the inclusion of baked egg in the diet accelerates the development of oral
354
tolerance.27-30 The mechanisms responsible for the reduced allergenicity of heat-treated (100° C,
355
5-60 min) EW, OVA and OM were investigated in BALB/c and C3H/HeJ mice. Results pointed
356
at the enhanced digestibility of heated OVA, which reduces its basophil activation capacity, and
357
the impaired absorption of immunologically active forms of the allergens through the intestinal
358
epithelium.24, 31-32 Similarly, in the case of milk whey proteins, heat treatment and subsequent
359
aggregation was reported to promote their uptake from Peyer patches rather than from intestinal
360
epithelium cells, what reduces their capacity to elicit anaphylactic reactions, but enhances their
361
immunogenicity.33 In this respect, while there seems to be a general agreement that severe
362
heating reduces the capacity of egg proteins to trigger allergic reactions, much less is known on
363
the effect of processing on their sensitizing potential.
364
It was reported that mice sensitized by i.p. administration to OVA heated at 70ºC for 10
365
min develop lower levels of OVA-specific IgE than mice sensitized to native OVA.34 However, 14 ACS Paragon Plus Environment
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366
in that study, the administration route may have masked the impact of digestion and absorption
367
in the gastrointestinal tract on the immunogenicity of the protein, since both the native and
368
denatured forms were equally recognized by the generated IgE antibodies. Likewise, native and
369
aggregated OVA (80ºC, 6 h) i.p. administered to mice generated similar levels of IgE antibodies
370
specific to either OVA form.35 Following oral administration, the physicochemical changes
371
caused by heat treatment on EW protein structure, and consequent decreased stability towards
372
proteolysis, possibly altered antigen processing and presentation by dendritic cells and T cell
373
priming, ultimately leading to the induction, in HEW-sensitized mice, of IgE over IgG1
374
antibodies with different specificities compared with those produced in EW-sensitized mice. In
375
addition, both structural changes brought about by heat treatment and increased degradation
376
during in vivo digestion could have impaired the ability of HEW for mast cell crosslinking and
377
activation, despite the IgE-antibodies generated showed high affinity towards HEW epitopes.
378
Our results indicate that mice sensitized to HEW by oral administration were more susceptible
379
to Th1 responses than those sensitized to EW, which points at a more equilibrated Th1/Th2
380
balance indicative of a lower sensitization status, an aspect also observed when heated OVA is
381
used for mice immunization through the i.p. route. 34-35 It has been described that different forms
382
of the same antigen can activate distinct patterns of T cell commitment in mice with
383
consequences in the generated antibodies. Thus, OVA chemically modified to give rise to high
384
molecular weight polymers shifts the activation of the OVA-reactive T cell repertoire towards a
385
Th1 phenotype in vivo, what correlates with the antibody responses36. It should be noted that, in
386
our work, spleen cells from HEW-sensitized mice produced the highest level of IFN-γ ex vivo
387
regardless of the stimulus. In fact EW, HEW and PEW induced the same cytokine responses on
388
cells from naïve or sensitized mice, which does not support that heat denatured egg proteins
389
intrinsically stimulate a Th1 profile over a Th2 one. 37
390
Unlike in the case of sensitization to HEW, sensitization to EW and PEW gave rise to an
391
allergic response likely mediated by both IgE and IgG1. Both isotypes can participate in
392
anaphylaxis, in mice, although the IgG1 pathway differs from the classical IgE pathway in that
393
it is mainly triggered by the binding of circulating antigen-IgG1 complexes to FcγRIII receptors 15 ACS Paragon Plus Environment
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394
on macrophages, instead of the crosslinking of IgE bound to FCεRI on basophils or mast cells.38
395
IgG1-dependent anaphylaxis in EW- and PEW-sensitized mice could have been facilitated by
396
the large excess of specific antibodies from the IgG1 isotype with respect to the IgE isotype,
397
particularly after the entry of the allergen into systemic circulation. Indeed, despite the fact that
398
the clinical relevance of non-IgE mediated anaphylaxis in humans is controversial, IgG-
399
mediated anaphylaxis can occur in the presence of large concentrations of antigens and of
400
specific IgG antibodies.
401
translated to a human situation, the fact that, in mice, IgE and IgG1 are Th2-driven isotypes
402
induced by IL-4,40 argues for a superior sensitization capacity of EW and PEW over HEW.
39
Thus, even if these results obtained in mice cannot be directly
403
EW and PEW induced equivalent IgE and IgG1 titers and the specificity of the generated
404
antibodies was also very similar. However, spleen cells of PEW-sensitized mice responded with
405
a higher secretion of IL-4, IL-5 and IL-10 to stimulation with either EW, HEW and PEW or to
406
the individual EW-proteins, which denotes a Th2-bias typical of the allergic status and suggests
407
that high pressure processing increased the sensitizing potential of EW proteins. Furthermore,
408
unlike challenge with EW, challenge of mice sensitized to EW with PEW provoked significant
409
temperature drops and mast cell degranulation as compared to naïve mice, suggesting that
410
limited denaturation caused by high hydrostatic pressure increased is eliciting potential. There is
411
some information on the impact of high hydrostatic pressure processing on the IgG- and IgE-
412
binding properties of food proteins4 but, to the best of our knowledge, there are no published
413
data on its effects on the induction of antibody responses and the acquisition of allergic
414
sensitization, or the elicitation of allergic reactions in vivo.
415
It is worth mentioning that, in EW-sensitized mice, the levels specific IgE were in the order
416
OM≥ OVA≥ LYS and there were similar concentrations of OVA-, OM- and LYS-specific IgG1,
417
despite these proteins are present in very different proportions in EW (OVA 54%, OM 11% and
418
LYS 3.5% w/w respectively). It is assumed that OM plays a predominant role in egg allergy,
419
but, in general terms, the contribution of the individual protein components and, in particular,
420
the influence of LYS, is insufficiently known.7 As compared with EW, LYS in HEW generated
421
a significantly lower IgE response, while LYS-specific IgE response in PEW was significantly 16 ACS Paragon Plus Environment
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422
higher. LYS is regarded as a thermolabile allergen.41 Unfolding of LYS becomes irreversible
423
when the temperature is above 80ºC, with α-helices being thermodynamically and kinetically
424
more stable than β-structures.42 However, under high hydrostatic pressure conditions (600 MPa,
425
30 min, 40ºC), partial and reversible unfolding of LYS occurs.22 Our results suggest that heat-
426
induced aggregation and enhanced flexibility brought about by high pressure decreased and
427
enhanced, respectively, LYS sensitizing potential, in support of previous findings showing that
428
LYS structure plays an important role in its immunogenicity. In fact, immunization of mice with
429
LYS derivatives of different conformational stability revealed that the least stable derivative
430
leads to the most potent Th2 response and IgE production, which was associated with a higher
431
susceptibility of the unfolded form to be processed by antigen presenting cells.43-44
432
In conclusion, extensive protein denaturation caused by heat treatment of EW not only
433
reduced its eliciting capacity but also its sensitizing capacity in a BALB/c model of egg allergy.
434
HEW stimulated IgE responses over IgG1 responses; however, given the involvement of both
435
isotypes in anaphylaxis in this animal model, the overall result is likely a lower degree of
436
sensitization, reinforced by the observation that HEW-sensitized mice were more prone to Th1
437
responses than EW-sensitized mice. Furthermore, HEW induced the production of antibodies
438
directed towards new epitopes exposed by heat treatment or released as a consequence of the
439
enhanced digestibility of the heated allergen. Conversely, partial denaturation caused by high
440
pressure treatment increased the ability of EW to stimulate Th2-biased responses and its
441
allergenic potential.
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442
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38.Finkelman, F. D. Anaphylaxis: Lessons from mouse models. J. Allergy Clin. Immunol. 2007, 120, 506-515. 39.Finkelman, F. D.; Khodoun, M. V.; Strait, R. Human IgE-independent systemic anaphylaxis. J. Allergy Clin. Immunol., 2016, 137, 1674-1680. 21 ACS Paragon Plus Environment
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40.Mestas, J.; Hughes, C. C. W. Of mice and not men: Differences between mouse and human immunology. J. Immunol. 2004, 172, 2731-2738. 41.Shin, M.; Han, Y.; Ahn, K. The influence of the time and temperature of heat treatment on the allergenicity of egg white proteins. Allergy Asthma Immunol. Res. 2013, 5, 96–101.
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42.Meersman, F.; Atilgan, C.; Miles, A. J.; Bader, R.; Shang, W.; Matagne, A.; Wallace, B. A.;
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Koch, M. H. J. Consistent picture of the reversible thermal unfolding of hen egg-white
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44.Peters, N. C.; Hamilton, D. H.; Bretscher, P. Analysis of cytokine-producing Th cells from
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Funding: This work was supported by MINECO (project AGL2014-59771R and contract of
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A.P-T), and MECD (through D.L-O contract).
567 568 569
Abbreviations used: CD, circular dichroism; CT, cholera toxin; EW, egg white; HEW,
570
heated egg white; i.p.., intraperitoneal; LYS, lysozyme; MCP-1, mast cell protease-1; OVA,
571
ovalbumin; OM, ovomucoid; PCA, passive cutaneous anaphylaxis; PEW, pressurized egg
572
white
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Table 1. Protein- specific IgE (ng/ml) and IgG1 (µg/ml) levels in the sera of mice sensitized to EW, HEW or PEW measured at 8th week (before challenge) by indirect ELISA. Naïve mice were used as controls. Data are expressed as means ± SEM (n= 5).
IgE (ng/ml)
IgG1 (µg/ml)
Sensitization group
Protein-specific Immunoglobulin
A-C a-c
EW HEW PEW Naïve EW HEW PEW Naïve
EW
OVA
OM
LYS
AB 1248.5 b ±216.7
AB 1407.7 b ±272.3
A 2121.9 a ±362.9
B 517.4 b ±62.9
A 2835.6 a ±316.0
A 3031.6 a ±309.7
A 2839.5 a ±588.3
B 38.0 c ±33.8
AB 1910.6 ab ±275.9
AB 1903.9 b ±318.8
A 2906.2 a ±439.1
B 958.0 a ±191.4
0.0 c ±0.0
0.0 c ±0.0
0.0 b ±0.0
0.0 c ±0.0
A 554.1 a ±45.4
B 276.0 a ±37.4
B 191.8 a ±34.7
B 314.6 a ±18.1
A 69.1 b ±9.5
A 76.3 b ±8.2
B 36.9 b ±10.2
C 1.8 b ±0.1
A 643.9 a ±50.8
B 371.9 a ±53.5
B 221.5 a ±48.1
B 364.9 a ±29.5
2.3 b ±0.6
2.5 b ±0.2
1.8 b ±0.1
1.7 b ±0.2
HEW
PEW
A 3674.8 a ±558.9 AB
0.0 b ±0.0 A 77.5 a
1901.0 a ±298.2 0.0 b ±0.0
±16.1 A 694.5 a ±75.5
1.3 b ±0.1
1.7 b ±0.2
Different uppercase superscript letters indicate significant differences (P< 0.05) within rows.
Different lowercase superscript letters indicate significant differences (P< 0.05) within columns for each antibody (IgE, IgG1).
24
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Figure captions Figure 1. SDS-PAGE patterns of EW, HEW and PEW, without and with β-mercaptoethanol (-βME and + β-ME respectively) (a). In vitro gastric (G, 20 and 60 min) and gastrointestinal (GI, 20 and 60 min) digests of EW, HEW and PEW with β-mercaptoethanol (b). MW: molecular weight marker; OVT: ovotransferrin; OVA: ovalbumin; OM: ovomucoid; LYS: lysozyme. Figure 2. Far-UV circular dichroism spectra of EW (−), HEW (--) and PEW (•••) at pH 7.0 and 20ºC. Figure 3. Body temperature (a), clinical sign scores (b), and serum concentrations of MCP-1 (c) in BALB/c mice sensitized to EW, HEW or PEW and challenged with these protein preparations. Oral challenges were followed by i.p. challenges 40 min apart. Values are expressed as means ± SEM (a, c) or medians (b). Different letters indicate statistically significant differences (P< 0.05) within orally or i.p. challenged animals (n= 5). Figure 4. Binding capacity of IgE from pooled serum samples of mice (n=5) sensitized to EW, HEW and PEW to each of these protein preparations determined by direct antibody capture ELISA (a). Competition of EW, HEW and PEW for the binding of IgE antibodies present in serum of EW- (b), HEW- (c) and PEW-sensitized mice (d) to biotin-labelled EW, HEW and PEW, respectively. Results are presented as means ± SEM and different letters indicate statistically significant differences (P< 0.05) (technical triplicates). Figure 5. Production of IFNγ, IL-4, IL-5 and IL-10 by splenocytes of mice sensitized to EW, HEW or PEW and stimulated with individual egg proteins (OVA, OM and LYS), as well as with these protein preparations. Data are expressed as means ± SEM (n= 5). Different lowercase letters indicate statistically significant differences (P< 0.05) within each sensitized group and different uppercase letters indicate statistically significant differences (P< 0.05) among different sensitization groups for the same stimulus.
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Supplementary Figure 1. Levels of IgE and IgG1 specific for EW, OVA, OM and LYS in sera from mice sensitized to EW (a), HEW (b), and PEW (c) at different days throughout the sensitization period determined by indirect ELISA. Data are expressed as means ± SEM (n=5). Supplementary Figure 2. Passive cutaneous anaphylaxis assay with unheated and heated pooled sera (n= 5) from HEW -sensitized mice. Data are expressed as means ± SEM (n=2). *indicates statistically significant differences between the responses induced by heated and non-heated sera (P< 0.05).
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TOC Graphic
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FIGURE 1 a)
b) - β-ME
EW
+ β-ME
MW EW HEW PEW EW HEW PEW
HEW
PEW
MW G20´ G60´ GI20´ GI60´ G20´ G60´ GI20´ GI60´ G20´ G60´ GI20´ GI60´
kDa 250 150 100 75
kDa 250 150 100 75
50
50
37
OVA 37
OVT
OM
25 20
25 20
15
15
10
10
LYS
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Ɵ (deg·cm2·cg-1)
FIGURE 2 2500 0 -2500 -5000 200
210
220
230
240
250
l (nm)
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FIGURE 3 Oral
a)
Temperature (ºC)
40
ab
a
38
i.p. a
a
ab a
b
b
b
ab
abc
a
a bc c
bc
36 34 32
EW Sensitization Challenge EW HEW PEW
HEW
PEW
EW HEW
EW PEW
Naïve
EW
HEW
PEW
EW HEW PEW
EW HEW
EW PEW
a
a
a
Naïve
b)
Clinical signs score
5
ab
4
ab
a
a
a ab
ab
3
b
bc 2
cd
c
1
d
c
0
Sensitization EW Challenge EW HEW PEW
HEW
PEW
EW HEW
EW PEW
EW
HEW
PEW
EW HEW PEW
EW HEW
EW PEW
Naïve
a
c)
MCP-1 pg/ml
30000
a
a
20000 ab
ab
10000
b 0 Sensitization Challenge
EW EW HEW PEW
b b HEW
PEW
EW HEW
EW PEW
ACS Paragon Plus Environment
Naïve
Naïve
Page 31 of 32
Journal of Agricultural and Food Chemistry
FIGURE 4 IgE 1.0 a
0.8
ab
0.6 b
0.4 0.2 0.0
c)
100
100
80
80 % B/B0
% B/B0
b)
EW
60 40
HEW
PEW
d)
60 40
REW 20 100
100
%B/B 0
80
2
4
6
0
Log10 [non-labelled protein]
60 40
40 20
2
4
6 EW Log10 [non-labelled protein] EW HEW EW HEW PEW HEW PEW PEW
20 0 0 2 4 Log10 [non-labelled protein] 0 2 4 6 Log10 [non-labelled protein] 2 4 6 Log10 [non-labelled protein]
0
40
REW
80 0
60
60
20
20
REW
100 80
% B/B0
Absorbance units (450 nm)
a)
6
ACS Paragon Plus Environment
0
2
4
6
Log10 [non-labelled protein]
Journal of Agricultural and Food Chemistry
Page 32 of 32
FIGURE 5
IFN (ng/mL)
20.0 15.0 10.0 5.0
B B B a a a B B B b b b
A A A a a a A A A b b b
B B a B ab B B b B cd c d
0 A a
1.5 IL 4 (ng/mL)
a AB a
1.0 0.5
a B ab ab
a a
B AB b b b
B ab
a
A b A c c
B ab
0
A A a A ab ab
IL 5 (ng/mL)
1.5 1.0 0.5
B B B B a a a B b b b
AB bc AB c c
B B B ab ab a
A bc c
A c
0 A
a
IL 10 (ng/mL)
6 4 2
B bc B B c c
B B B a ab a
B ab
B B B a a ab
A a
A b
A a
A A b b
B B b b
0
ACS Paragon Plus Environment
PEW
HEW
EW
LYS
OM
OVA
PEW
PEW HEW
EW
LYS
OM
OVA
PEW
HEW
EW
LYS
OM
OVA
Cell stimulus
HEW
EW
Sensitization