Egg-white-peptide-based-immunotherapy-enhances-vitamin-A 2019 Journal-of-Fun

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Egg white peptide-based immunotherapy enhances vitamin A metabolism

and induces RORγt

regulatory T cells

Daniel Lozano-Ojalvo

, Mónica Martínez-Blanco

, Leticia Pérez-Rodríguez, Elena Molina,

Carmen Peláez, Teresa Requena, Rosina López-Fandiño

⁎

Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Nicolás Cabrera 9, 28049 Madrid, Spain

ARTICLE INFO

Keywords:

Food allergy

Egg-peptide immunotherapy

Microbiota

Retinoic acid

RORγt

Regulatory T cells

ABSTRACT

This study investigates the mechanism by which food peptides are more eﬀective than intact allergens in pro-

viding desensitization against food allergy. BALB/c mice sensitized to egg white (EW) were subjected to oral

immunotherapy (OIT) with intact and pepsin-hydrolysed EW (EP). Treatment with EP was superior to that with

EW in terms of reduction of anaphylaxis and levels of speciﬁc antibodies. OIT with EP, but not with EW,

modulated the microbiota by restoring the levels of some members of the order Clostridiales (clusters IV and

XIVa) that were aﬀected by sensitization. Mice treated with EP exhibited upregulated intestinal expression of

Il22 and Muc2, which encode for factors that contribute to reinforce the epithelial barrier function, as well as

Aldh1a1,Aldh1a2,Csf2 and Tfgb1, that take part in the conversion of vitamin A into retinoic acid. Tolerance

induced by EP paralleled the development of Foxp3

cells that simultaneously expressed RORγt.

1. Introduction

In case of food allergy, desensitization can be induced through the

gradual administration of increasing amounts of allergen with rates of

success around 70% (Tordesillas, Berin, & Sampson, 2017). However,

oral immunotherapy (OIT) using intact allergens has disadvantages

derived from the facts that it often leads to the appearance of adverse

reactions and there is little evidence for the establishment of long

lasting tolerance (Berin & Shreﬄer, 2016). Previous results demon-

strated that treatment with hydrolysed ovalbumin, but not with the

intact protein, signiﬁcantly attenuates anaphylaxis in BALB/c mice

sensitized to egg white (EW) following challenge with the allergen, a

protection that is maintained at least for 3 weeks after discontinuation

of the treatment along with an increase of regulatory T (Treg) cells

(Lozano-Ojalvo, Pérez-Rodríguez, Pablos-Tanarro, Molina, & López-

Fandiño, 2017). The lack of IgE-binding activity of hydrolysed oval-

bumin is likely to increase eﬃcacy of OIT, since stimulation of mast

cells releases Th2 cytokines that interfere with the diﬀerentiation of the

Treg cells through the inhibition of Foxp3 (Burton et al., 2014). Fur-

thermore, the hydrolysate may contain speciﬁc peptides active in the

promotion of Treg cells, as we detected an increase in the mesenteric

lymph node (MLN) population of CD103

CD11b

dendritic cells

(DCs) (Lozano-Ojalvo et al., 2017). CD103

DCs are regarded as tol-

erogenic by virtue of the production of TGF-β and retinoic acid (RA)

(Coombes et al., 2007), and it is known that dietary antigens diﬀeren-

tially inﬂuence the generation of this subset (Kim, Hong, et al., 2016).

An unexpected result was the overexpression, in the duodenum of

the animals treated with hydrolysed ovalbumin, together with Foxp3

and Il10, of Rorc and Il17, which encode, respectively, for the tran-

scription factor RORɣt and distinctive cytokine from Th17 cells

(Lozano-Ojalvo et al., 2017). Ohnmacht et al. (2015) showed that the

intestinal microbiota stimulates the expression of RORɣt in colonic Treg

cells and that this inhibits Th2 cells, avoiding the production of IL-4 and

IgE. Indeed, the intestinal microbiota is a major factor in the develop-

ment of innate and acquired immune responses and in the susceptibility

to food allergy (Huang et al., 2017), although it is currently unknown if

Foxp3

RORɣt

cells play a role in the acquisition of tolerance

mediated by OIT. These observations, together with studies indicating

that hydrolysed egg proteins can exert positive eﬀects on intestinal

dysbiosis (Requena et al., 2017), suggest an interrelation between

https://doi.org/10.1016/j.jﬀ.2018.11.012

Received 27 September 2018; Received in revised form 5 November 2018; Accepted 5 November 2018

Abbreviations: CT, cholera toxin; DC, dendritic cell; EP, pepsin-hydrolyzed egg white; EW, egg white; MLN, mesenteric lymph node; mMCP-1, mouse mast cell

protease-1; OIT, oral immunotherapy; qPCR, quantitative PCR; RA, retinoic acid; SCFA, short chain fatty acid; Treg cells, regulatory T cells

⁎

Corresponding author.

E-mail address: [email protected] (R. López-Fandiño).

These authors contributed equally to this work.

Journal of Functional Foods 52 (2019) 204–211

Available online 16 November 2018

regulatory functions in the small and large intestine, provided both by

bioactive peptides and commensal microorganisms, which might be

involved in desensitization to food allergens.

In order to understand the mechanism by which food peptides

confer protection against established food allergy, we subjected mice

sensitized to EW to OIT with intact and hydrolysed EW. Changes in the

microbiota, generation of barrier-protective responses in the small and

large intestine and stimulation of a regulatory environment in non-

lymphoid and lymphoid tissues were studied. The results showed that

OIT with peptides resolved allergic symptoms and modulated the mi-

crobial alterations that accompanied sensitization. The health beneﬁts

of peptide OIT were associated to vitamin A metabolism and develop-

ment of innate and adaptive cells that depend on RORγt for their

transcriptional regulation.

2. Materials and methods

2.1. Materials

Whole EW hydrolysed with pepsin (EP) was used as OIT, instead of

hydrolysed ovalbumin, because of its lower cost and ease of production

for larger scale uses. EW was obtained from fresh hen eggs. Hydrolysis

was conducted with 172 U/mg of protein of porcine pepsin (EC

3.4.23.1, 3440 U/mg, Sigma-Aldrich, St. Louis, MO, USA), at pH 1.5

and 37 °C for 24 h. The hydrolysate was neutralized to pH 7.0, heated at

95 °C for 15 min, centrifuged (5000g, 4 °C, 10 min) and lyophilized. The

absence of lipopolysaccharide was conﬁrmed (Pierce®LAL, Thermo

scientiﬁc, Waltham, USA) and the protein content was analysed by the

Kjeldahl method.

2.2. Protocols in mice

Six week-old female BALB/c mice (Charles River Laboratories, Saint

Germain sur ĺArbresle, Rhône, France) were distributed in 5 groups (5

per group). Three groups were sensitized to EW by the intragastric

administration of 5 mg of EW on a protein basis and 10 μg of cholera

toxin (CT, List Biologicals, Campbell, CA, USA), during 3 successive

days on the ﬁrst week and once per week for the subsequent 5 weeks (Li

et al., 2000). Sham-sensitized mice received CT and naïve mice just

received PBS. One week after, 2 groups of EW-sensitized mice were

administered intragastrically the amount equivalent to 5 mg of protein

of EW or EP, 3 times per week during 3 weeks. Mice from the other EW-

sensitized group, the sham-sensitized group and the naïve group were

administered PBS. Three days after, mice from all groups were in-

tragastrically challenged with 50 mg (on a protein basis) of EW. Thirty

min apart, anaphylaxis was evaluated by scoring clinical signs and

rectal temperature and 3 h later, mice were euthanized by CO

in-

halation (Pablos-Tanarro, Lozano-Ojalvo, Molina, & López-Fandiño,

2018).

In order to assess whether oral treatment with EW or EP could have

any direct impact on the caecal microbiota and its metabolism,

12 week-old naïve mice distributed in 3 groups (5 per group) were

administered PBS or 5 mg of protein of EW or EP, 3 times per week

during 3 weeks, as above, and euthanized 3 days later.

Serum levels of EW-speciﬁc IgE and IgG1, and mouse mast cell

protease-1 (mMCP-1) were quantiﬁed by ELISA (Pablos-Tanarro et al.,

2018). The caecal content was removed and stored at -80 °C. Duodenum

and colon segments and MLNs were preserved in storage buﬀer (Ma-

cherey-Nagel Gmbh & Co., Düren, Germany) at -80 °C for gene ex-

pression analyses. Spleen cells were isolated for ﬂow cytometry as

previously described (Lozano-Ojalvo et al., 2017).

All protocols involving animals followed the European legislation

(Directive 2010/63/EU) and were approved by Comunidad de Madrid

(Ref PROEX 089/15).

2.3. Microbiological analyses

The caecal content was suspended in 0.1% peptone solution with

0.85% NaCl and centrifuged (10000g, 4 °C, 5 min). Pellets were used for

DNA extraction (Moles et al., 2013), and supernatants for short chain

fatty acid (SCFA) analysis (Requena et al., 2017). Quantitative PCR

(qPCR) was performed using SYBR green methodology in a ViiA7 Real-

Time PCR System (Life Technologies, Carlsbad, CA, USA), as shown in

Supplementary Table S1. SCFAs were analysed by HPLC (Jasco, Tokyo,

Japan) with a Rezex ROA column (Phenomenex, Macclesﬁeld, UK) and

detection at 210 nm (Sanz et al., 2005).

2.4. Gene expression

Total RNA from duodenum, colon and MLNs was isolated using

NucleoSpin RNA Kit (Macherey-Nagel Gmbh & Co., Düren, Germany)

and cDNA was synthetized with PrimeScript RT kit (TaKaRa Bio Inc.,

Shiga, Japan). Conditions for qPCR are shown in Supplementary Table

S2. Relative gene expression was calculated by normalizing data to the

expression of the reference gene Actb, using either the sham-sensitized

or the naïve group as calibrators (Livak & Schmittgen, 2001).

2.5. Flow cytometry of T cells

Isolated splenocytes were recovered in PBS containing 2% fetal

bovine serum and 1 mM EDTA. Fc receptors were blocked using anti-

CD16/CD32 (clone 2.4G2, BD Biosciences) and live cells determined

with LIVE/DEAD®Kit (Thermo Fisher Scientiﬁc, Walthman, USA).

Samples were stained with the antibodies listed in Supplementary Table

S3 and analysed as shown in Supplementary Figure S1. Cells were ac-

quired with a Gallios ﬂow cytometer (Beckman Coulter, Krefeld,

Germany) and analyses were performed with FlowJo for windows

(version 7.6.5).

2.6. Statistical analyses

Results are presented as means ± SEM, except for clinical signs,

which are expressed as medians. Diﬀerences were determined by one-

way ANOVA followed by Tukey post-hoc test, except for clinical signs

and relative gene expression data, which were evaluated by Mann-

Whitney Utest. P< 0.05 was considered statistically signiﬁcant.

Analyses were performed with GraphPad Prism v5 (GraphPad Software,

San Diego, USA).

3. Results

3.1. Immunotherapy with peptides promotes oral tolerance and reverses

microbial alterations in mice sensitized to egg white with the aid of cholera

toxin

Administration of EP to EW-sensitized mice prevented anaphylaxis

and inhibited the release of mast cell mediators (mMCP-1) upon in-

tragastric challenge with the allergen (Supplementary Figure S2).

However, mice administered EW experienced similar clinical signs and

body temperature drops than the non-treated animals, although serum

levels of mMCP-1 were signiﬁcantly lower. The improved condition of

mice treated with EP was accompanied by a reduction in the con-

centration of circulating EW-speciﬁc IgE and IgG1 (Supplementary

Figure S3).

Sensitization to EW using CT as adjuvant gave rise to decreases in

the abundance of total caecal bacteria, Clostridium leptum,

Ruminococcus,Roseburia and Blautia coccoides/Eubacterium rectale,

while Akkermansia was enhanced (Fig. 1). Administration of CT alone

decreased Enterobacteriaceae and Bacteroides and increased Biﬁdo-

bacterium. In this respect, even if CT is broadly used as a Th2-driving

agent to induce sensitization in mice and it has been recognized that the

D. Lozano-Ojalvo et al. Journal of Functional Foods 52 (2019) 204–211

205

microbiota plays a role in its adjuvant activity (Kim, Kim, et al., 2016),

to the best of our knowledge, the microbiota proﬁle characteristic of CT

administration or of CT-induced sensitization to co-administered anti-

gens has not been deﬁned to date.

Interestingly, OIT with EP restored the amount of total bacteria, C.

leptum, Ruminococcus, and B. coccoides/E. rectale to the levels of naïve

mice, while those of Roseburia and Akkermansia remained altered

(Fig. 1). Overall, treatment with EW did not modify the caecal bacterial

composition of the sensitized animals. The caecal concentrations of the

SCFAs, acetate, propionate and butyrate were analysed in all mice

groups and a signiﬁcant drop in propionate content was observed in the

non-treated animals, that regained the former state following EP and

EW treatments (Fig. 2). In order to ﬁnd out whether changes in the

microbiota were due to a direct eﬀect of peptides reaching the large

intestine, naïve mice were similarly administered EP or EW for an

equivalent time period, but no diﬀerences in caecal bacteria were de-

tected with respect to naïve mice given PBS (Supplementary Figure S4).

Therefore, OIT with peptides helped to re-establish the altered

Fig. 1. Microbial groups in caecal contents collected after sacriﬁce. Mice were sham-sensitized (CT) or sensitized to EW and treated with PBS, EW or EP. Naïve mice

are also shown for comparison. Data are expressed as means ± SEM (n = 5). * P< 0.05, ** P< 0.01, *** P< 0.001 vs naïve mice.

Fig. 2. SCFAs acetate, propionate and butyrate in caecal contents collected after sacriﬁce. Mice were sham-sensitized (CT) or sensitized to EW and treated with PBS,

EW or EP. Naïve mice are also shown for comparison. Data are expressed as means ± SEM (n = 5). ** P< 0.01 vs naïve mice.

D. Lozano-Ojalvo et al. Journal of Functional Foods 52 (2019) 204–211

206

intestinal microbial proﬁle generated by CT-induced sensitization by

resolving the allergic status rather than exerting a direct inﬂuence on

the microbiota.

3.2. Protection from anaphylaxis is accompanied by changes in the

expression of genes associated with epithelial integrity in the small and large

intestine

We next evaluated the gene expression of intestinal barrier para-

meters that act as markers of mucosal protection mechanisms and

permeability, such as the cytokine IL-22, considered a key component

in the crosstalk among microbiota, intestinal epithelium and immune

cells (Schreiber, Arasteh, & Lawley, 2015); its soluble-secreted receptor,

IL-22 binding protein (termed IL-22BP or IL-22RA2), that speciﬁcally

binds to IL-22 and inhibits its biological eﬀects (Martin et al., 2014);

Muc2, a highly glycosylated mucin, which is the major constituent of

the mucus layer in the colon (Johansson, Larsson, & Hansson, 2011);

and the tight junction molecules claudin-2 and zonula occludens-1

(Volynets et al., 2016). Il22,Muc2 and Cldn2 were overexpressed

(P< 0.05) in the intestinal tissues of mice sensitized to EW, and

Il22ra2 was upregulated in the duodenum (Fig. 3). Expression of Il22

was reduced by OIT with EW, but treatment with EP markedly upre-

gulated Il22 and Il22ra2 in the duodenum and Muc2 in the colon

(Fig. 3). Expression of Cldn2 and Tjp1 was not aﬀected, except for a

tendency (P= 0.2) of EP to enhance Tjp1 in the colon (Fig. 3). Ad-

ministration of EW or EP to naïve mice did not change the expression of

Il22 or Muc2 (not shown). These results denote an inﬂuence of OIT with

peptides on factors that contribute to reinforce epithelial barrier func-

tion.

3.3. Tolerance concurs with the expansion of Foxp3

RORγt

cells

OIT with both EW and EP decreased the expression of Gata3, which

was signiﬁcantly (P< 0.05) upregulated in mice sensitized to EW, at

the small and large intestinal level and in the MLNs (Fig. 4). As com-

pared to EW, treatment with EP distinctively upregulated Foxp3 in the

duodenum, and also enhanced Rorc expression in the duodenum, colon

and MLNs. Expression of Il10 was unchanged in non-lymphoid tissues,

albeit it increased in the MLNs as a result of the treatments, that also

enhanced Il17 expression in the duodenum and colon (Fig. 4).

Analysis of CD4

T cell populations in the spleen showed that OIT

with EP decreased the percentage of activated Th1 and Th2 cells and

increased that of Foxp3

Treg cells (Fig. 5). The percentage of RORγt

T cells was also enhanced, as well as that of Foxp3

RORγt

T cells. In

fact, the proportion of Foxp3

cells that co-expressed RORγt in the

spleens of mice treated with EP was signiﬁcantly higher than that of

untreated mice or mice treated with EW (Fig. 5). This observation, to-

gether with the enhanced expression of Rorc at the intestinal level

brought about by EP, strongly suggests that the induction of double

positive Foxp3

RORγt

cells played a role in the therapeutic eﬀect of

EP.

3.4. Immunotherapy with peptides induces intestinal expression of enzymes

that metabolize vitamin A into retinoic acid

Several reports have indicated that the vitamin A metabolite RA

promotes the generation of Treg and RORγt

cells (Mucida et al., 2007;

Ohnmacht et al., 2015). Therefore, we studied the expression of

Aldh1a1 and Aldh1a2 genes, which encode for the retinaldehyde de-

hydrogenases RALDH1 and RALDH2, the major isoforms expressed by

mouse intestinal epithelial cells and DCs, respectively, that oxide retinal

to RA (Hall, Grainger, Spencer, & Belkaid, 2011). Expression of Aldh1a1

and Aldh1a2 was increased in the duodenum and colon of EP-, but not

of EW-treated mice, and expression of Aldh1a2 was also enhanced in

the MLNs of the former (Fig. 6). Expression of Tfgb1 followed a similar

trend, while that of Csf2, which encodes for GM-CSF, that induces DCs

to express Aldh1a2 (Yokota et al., 2009),was signiﬁcantly increased in

the colon and MLNs (Fig. 6). RA signalling has been shown to promote

TGF-β and IL-6 production in DCs (Feng, Cong, Qin, Benveniste, &

Elson, 2010). However, expression of Il6, markedly enhanced in the

duodenum and MLNs of EW-sensitized and challenged mice

(P< 0.01), was decreased in mice subjected to OIT with either EW or

EP, likely reﬂecting the neutralization of allergic inﬂammatory re-

sponses (Fig. 6). Noteworthy, administration of EW and EP to naïve

mice did not change the expression of Aldh1a1,Aldh1a2,Tfgb1 or Il6

(not shown), implying the absence of eﬀects on vitamin A metabolism

Fig. 3. Relative gene expression of Il22, Il22ra2,

Muc2, Cldn2 and Tjp1 determined in the duodenum

(DD) and colon (CL). Mice were sensitized to EW

and treated with PBS, EW or EP. The sham-sensi-

tized (CT) group, used as calibrator, is represented

as a discontinuous line in the ﬁgure. Data are ex-

pressed as means ± SEM (n = 5). * P< 0.05, **

P< 0.01, *** P< 0.001 vs sensitized mice treated

with PBS.

D. Lozano-Ojalvo et al. Journal of Functional Foods 52 (2019) 204–211

207

under homeostatic conditions. These results suggest that OIT with

peptides could induce Foxp3

RORγt

cells through a RA- and TGF-β-

dependent mechanism.

4. Discussion

This study conﬁrms that OIT with hydrolysed allergens is more ef-

fective than that with the intact proteins. The distinctive properties of

peptide OIT are linked to the restoration of microbial alterations, the

induction of factors that contribute to reinforce the intestinal barrier

function, such as IL-22 and Muc2, the generation of Foxp3

RORγt

Treg cells and the promotion of enzymes that catalyse the production of

RA.

Noval Rivas et al. (2013) found, in mice genetically susceptible to

develop food allergy, a characteristic microbiota that transmits this

susceptibility when transferred to germ-free mice. In agreement with

these authors, our results, in an adjuvant-induced mouse model of food

allergy, show that an allergic status arising from an exaggerated Th2

response was associated with microbial alterations. Reciprocally, the re-

establishment of tolerance, by the induction of a regulatory environ-

ment through peptide OIT, led to measurable changes in the micro-

biota.

In our mouse model, allergy to EW was associated to decreased

levels of some members of the order Clostridiales (clusters IV and

XIVa), in parallel with an increase in bacteria from the genus

Akkermansia. Clostridiales are considered beneﬁcial in the avoidance of

food allergy (Huang et al., 2017). Atarashi et al. (2011 and 2013)

identiﬁed Clostridium species belonging to clusters IV, XIVa and XVIII as

inductors of Foxp3

Treg cells in the colonic lamina propria. On the

other hand, Clostridia-containing microbiota stimulates the intestinal

production of IL-22, which favours epithelial integrity and promotes the

expression of antimicrobial peptides and mucus (Stefka et al., 2014).

However, it is unlikely that the induction of factors that contribute to

barrier function by Clostridia colonization provide protection against

food allergy, because neutralization of IL-22 does not increase the

susceptibility of mice to sensitization (Stefka et al., 2014).

In fact, in our study, Il22 was overexpressed in the intestine of

sensitized, not-treated mice. IL-22 induces innate responses that involve

not only anti-inﬂammatory, but also inﬂammatory eﬀects depending on

the tissue and the cytokine environment (Zenewicz & Flawell, 2011).

Thus, IL-22 is associated with the severity of asthma, allergic rhinitis

and atopic dermatitis, but almost no studies have investigated its role in

the pathogenesis of food allergy, even if it is amply produced by several

innate and adaptive immune cells in the small intestine, where the

balance between allergy and tolerance is sustained (Souwer, Szegedi,

Kapsenberg, & de Jong, 2010). It is known that IL-22 collaborates with

Th2 cytokines, such as IL-13 and IL-4, in the protection against in-

testinal helminth infections through goblet cell hyperplasia and en-

hanced expression of mucins (Turner, Stockinger, & Helmby, 2013).

Given the implication of several signalling pathways designed for pro-

tection from parasites in allergic inﬂammation (Ruiter & Shreﬄer,

2012), it can be reasoned that overexpression of Il22 was integrated

with the antigen-speciﬁc responses induced by CT that led to allergic

sensitization or formed part of tissue repair mechanisms activated by

the exacerbated Th2 immune milieu or by allergen challenge. Indeed,

Muc2 was overexpressed in the duodenum of sensitized mice, but

mainly in the colon, particularly rich in IL-22 target cells (Nagalakshmi,

Rascle, Zurawski, Menon, & de Waal Malefyt, 2004), and this concurred

with a high intestinal abundance of mucin-degrading bacteria from the

genus Akkermansia, whose colonization is promoted by mucus hy-

persecretion (Belzer & de Vos, 2012; Everard et al., 2013). IL-22 also

modiﬁes intestinal permeability through the induction of the expression

of Cldn2 (Wang, Mumm, Herbst, Kolbeck, & Wang, 2017), which was

upregulated in EW-sensitized and challenged mice in our study.

OIT with EP further upregulated the expression of Il22 in the duo-

denum and Muc2 in the colon. Although the reason for this behaviour

remains to be elucidated, it is noteworthy that a variety of RORγt

cells

from innate origin, mainly innate lymphoid cells type 3 (ILC3), found in

intestinal tissues and lymphoid follicles and whose diﬀerentiation de-

pends on RA, express high amounts of IL-22 (Ohnmacht, 2016). RA is

also a potent inducer of IL-22RA2 in DCs, that participates in the reg-

ulation of the deleterious eﬀects of IL-22 (Martin et al., 2014) and,

Fig. 4. Relative gene expression of Gata3,Foxp3,Rorc,Il10 and Il17 determined in the duodenum (DD), colon (CL) and mesenteric lymph nodes (MLNs). Mice were

sensitized to EW and treated with PBS, EW or EP. The sham-sensitized (CT) group, used as calibrator, is represented as a discontinuous line in the ﬁgure. Data are