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
1
, Mónica Martínez-Blanco
1
, 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).
1
These authors contributed equally to this work.
Journal of Functional Foods 52 (2019) 204–211
Available online 16 November 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
T

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
2
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
expressed as means ± SEM (n = 5). * P< 0.05, ** P< 0.01, *** P< 0.001 vs sensitized mice treated with PBS.
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