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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]

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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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1 ACS Paragon Plus Environment


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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

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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

95

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

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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

114

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

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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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groups. 10 ACS Paragon Plus Environment

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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

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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

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the immunization using non-heated serum, denoting that IgE was biologically functional and

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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

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EW-, HEW- and PEW-sensitized mice to EW subjected to different processing methods using a

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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

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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

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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,

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the observation that, despite allergy in HEW-sensitized mice was mainly IgE-mediated and

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HEW-specific IgE displayed higher affinity for HEW than for EW (Supplementary Fig. 2 and

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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

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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).

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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

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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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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.



Page 18 of 32

442

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443

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Immunol. 2011, 127, 990–997 e2.

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26.Bernard, H.; Drumare, M. F.; Guillon, B.; Paty, E.; Scheinmann, P.; Wal, J. M.

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observations. J. Allergy Clin. Immunol., 2008, 122, 414-415.

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31.Peng, H. J.; Chang, Z.N.; Tsai, L.C.; Su, S.N.; Shen, H.D.; Chang C.H. Heat denaturation of

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Jarolim, E.; Mayer, L. Pasteurization of milk proteins promotes allergic sensitization by

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enhancing uptake through Peyer's patches. Allergy, 2008, 63, 882-890.

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34.Golias, J.; Schwarzer, M.; Wallner, M.; Kverka, M.; Kozakova, H.; Srutkova, D.; Klimesova

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K.; Sotkovsky, P.; Palova-Jelinkova, L.; Ferreira, F.; Tuckova, L. Heat-induced structural

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35.Claude, M.; Lupi, R.; Bouchaud, G.; Bodinier, M.; Brossard, C.; Denery-Papini, S. The

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thermal aggregation of ovalbumin as large particles decreases its allergenicity for egg

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36.Gieni, R. S.; Yang, X.; Kelso, A.; Hayglass, K. T. Limiting dilution analysis of CD4 T-cell

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37.Rupa, P.; Schnarr, L.; Mine, Y. Effect of heat denaturation of egg white proteins ovalbumin

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production. J. Funct. Foods, 2015, 18, 28-34.

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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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lysozyme from experiment and molecular dynamics. Biophys J. 2010, 99, 2255–2263.

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43.So, T.; Ito, H.; Hirata, M.; Ueda, T.; Imoto, T. (2001). Contribution of conformational

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stability of hen lysozyme to induction of type 2 T-helper immune responses. Immunology,

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2001, 104, 259-268.

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44.Peters, N. C.; Hamilton, D. H.; Bretscher, P. Analysis of cytokine-producing Th cells from

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hen egg lysozyme-immunized mice reveals large numbers specific for "cryptic" peptides and

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different repertoires among different Th populations. Allergy, 2011, 41, 20–28.


Page 23 of 32


565

Funding: This work was supported by MINECO (project AGL2014-59771R and contract of

566

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



Page 24 of 32

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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Page 25 of 32


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.



Page 26 of 32

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).


Page 27 of 32


TOC Graphic



Page 28 of 32

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


Page 29 of 32


Ɵ (deg·cm2·cg-1)

FIGURE 2 2500 0 -2500 -5000 200

210

220

230

240

250

l (nm)



Page 30 of 32

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


Naïve

Naïve

Page 31 of 32


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


0

2

4

6

Log10 [non-labelled protein]


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


PEW

HEW

EW

LYS

OM

OVA

PEW

PEW HEW

EW

LYS

OM

OVA

PEW

HEW

EW

LYS

OM

OVA

Cell stimulus

HEW

EW

Sensitization

c mice

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