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

Journal of Functional Foods 52 (2019) 204–211
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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
T
Daniel Lozano-Ojalvo1, Mónica Martínez-Blanco1, 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
ABSTRACT
Keywords:
Food allergy
Egg-peptide immunotherapy
Microbiota
Retinoic acid
RORγt
Regulatory T cells
This study investigates the mechanism by which food peptides are more effective than intact allergens in providing 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 demonstrated 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, & LópezFandiño, 2017). The lack of IgE-binding activity of hydrolysed ovalbumin 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). Furthermore, 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 tolerogenic by virtue of the production of TGF-β and retinoic acid (RA)
(Coombes et al., 2007), and it is known that dietary antigens differentially 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 transcription 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 development 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
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.
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
Available online 16 November 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 52 (2019) 204–211
D. Lozano-Ojalvo et al.
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 nonlymphoid and lymphoid tissues were studied. The results showed that
OIT with peptides resolved allergic symptoms and modulated the microbial alterations that accompanied sensitization. The health benefits
of peptide OIT were associated to vitamin A metabolism and development of innate and adaptive cells that depend on RORγt for their
transcriptional regulation.
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 RealTime 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. 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.
2.5. 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 antiCD16/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 acquired with a Gallios flow cytometer (Beckman Coulter, Krefeld,
Germany) and analyses were performed with FlowJo for windows
(version 7.6.5).
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 EWsensitized group, the sham-sensitized group and the naïve group were
administered PBS. Three days after, mice from all groups were intragastrically 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 CO2 inhalation (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 (Macherey-Nagel Gmbh & Co., Düren, Germany) at -80 °C for gene expression 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).
2.6. Statistical analyses
Results are presented as means ± SEM, except for clinical signs,
which are expressed as medians. Differences were determined by oneway ANOVA followed by Tukey post-hoc test, except for clinical signs
and relative gene expression data, which were evaluated by MannWhitney U test. 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 intragastric 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 concentration 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 Bifidobacterium. 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
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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.
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 antigens 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 detected with respect to naïve mice given PBS (Supplementary Figure S4).
Therefore, OIT with peptides helped to re-establish the altered
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.
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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-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.
intestinal microbial profile generated by CT-induced sensitization by
resolving the allergic status rather than exerting a direct influence on
the microbiota.
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, together 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.
3.2. 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 parameters 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 upregulated 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). Administration 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 function.
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 dehydrogenases 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 responses (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
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 compared 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,
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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.
under homeostatic conditions. These results suggest that OIT with
peptides could induce Foxp3+ RORγt+ cells through a RA- and TGF-βdependent mechanism.
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 intestinal helminth infections through goblet cell hyperplasia and enhanced expression of mucins (Turner, Stockinger, & Helmby, 2013).
Given the implication of several signalling pathways designed for protection 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 hypersecretion (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 duodenum 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 depends 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 regulation of the deleterious effects of IL-22 (Martin et al., 2014) and,
4. Discussion
This study confirms that OIT with hydrolysed allergens is more effective 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 reestablishment of tolerance, by the induction of a regulatory environment through peptide OIT, led to measurable changes in the microbiota.
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).
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Fig. 5. Activated Th1, Th2, Foxp3+, RORγt+ and double positive Foxp3+ RORγt+ cells within the CD4+ population, and RORγt+ cells within the CD4+ Foxp3+
population in spleens. Data are expressed as means ± SEM (n = 5). Mice were sham-sensitized (CT) or sensitized to EW and treated with PBS, EW or EP. Data are
expressed as means ± SEM (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001 vs sensitized mice treated with PBS.
interestingly, treatment with EP enhanced the expression of Il22ra2.
Since treatment with EP upregulated RA metabolism, as judged by its
effect on Aldh1a1 and Aldh1a2 genes, it could have influenced ILC3s,
which, in turn, are producers of GM-CSF, that contributes to enhance
RA signalling (Mortha et al., 2014). Furthermore, a positive relationship between RA and the expression mucin and tight junction genes has
been established (Amit-Romach, Uni, Cheled, Berkovich, & Reifen,
2009; Osanai et al., 2007). Overall, our results indicate that EP may
exert protective barrier functions and provide tolerizing signals to DCs
via the activation of RALDH enzymes.
The reshaping of the immune response induced by EP was likely to
occur, at least partially, through the enhancement of regulatory responses, as shown by the increased expression of Foxp3 in intestinal
tissues and MLNs. Expression of Rorc and Il17 was also augmented and,
indeed, treatment with EP, but not with EW, increased the percentage
of splenic CD4+ cells that simultaneously borne Foxp3 and RORγt.
Despite the opposite primary functions of Foxp3 and RORγt, T cells that
co-express both transcription factors and produce IL-17 are found in
different mouse tissues and human PBMCs (Ayyoub et al., 2009;
Lochner et al., 2008; Zhou et al., 2008). These cells were proved to be
functionally suppressive, inhibiting in vitro proliferation of activated
CD4+ effector T cells and constraining inflammatory responses in vivo,
and to constitute a distinct, stable cell lineage, rather than representing
an intermediate subset during Treg and Th17 differentiation (Lochner
et al., 2008; Sefik et al., 2015; Yang et al., 2016). They are generated by
a specific, but wide, diversity of bacterial species, including Clostridium
(Geva-Zatorsky et al., 2017; Ohnmacht et al., 2015; Sefik et al., 2015).
While the underlying mechanisms are not fully clear, involvement of
antigens and metabolites derived from the gut microbiota, such as
SCFAs, mostly butyric acid, which stimulates epithelial production of
RA, has been proposed (Ohnmacht et al., 2015; Schilderink et al.,
2016), although, according to Sefik et al. (2015), there is no correlation
between any SCFA and RORγt frequency or other Treg parameters.
Furthermore, dietary antigens were shown to induce the expression of
RORγt and Foxp3 in naïve CD4+ T cells specific for that antigen
transferred to mice (Kim, Hong, et al., 2016; Ohnmacht et al., 2015).
Nevertheless, the induction of double positive cells by OIT with protein
hydrolysates has not been appreciated to date.
Unlike the hydrolysed proteins, intact EW was not able to generate
Foxp3+ RORγt+ CD4+ cells. The observation that OIT with EP was
superior, in terms of reduction of specific antibody levels, clinical signs
and release of mast cell mediators, suggests that these cells may be
crucial in the generation of tolerance to dietary antigens, in a way similar to the generation of tolerance to the intestinal microbiota.
Whereas it is not possible to discern to what extent changes in the
microbiota arising from OIT with EP were in part responsible for the
expansion of double positive cells, it is noteworthy that mice treated
with EP presented an enhanced expression of Aldh1a1, Aldh1a2 and
Tfgb1, not only in the colon and MLNs, but also in the duodenum, which
supports the heaviest load of peptides. This points at the peptides
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Fig. 6. Relative gene expression of Aldh1a1, Aldh1a2, csf2, Tgfb1 and Il6 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.
present in EP as the main cause of RALDH enzyme regulation, even if
the impact of a Th2-dominated environment deserves further investigation, in view of the absence of similar effects when EP was administered to naïve mice. RA is a characteristic component of local
milieus that preferentially drive the induction and expansion of
RORγt+ T reg cells over that of Th17 cells in vivo and in vitro (Lochner
et al., 2008; Ohnmacht et al., 2015). TGF-β can also induce the expression, together with Foxp3, of RORγt on CD4+ T cells, depending on
a tight balance regulated by the concentration of pro-inflammatory
cytokines, such as IL-6, IL-21 and IL-23 (Lochner et al., 2008; Zhou
et al., 2008). In this respect, it is remarkable that OIT, either with EW or
EP, reduced the exacerbated Il6 expression in the duodenum and MLNs
of sensitized mice. Furthermore, RA can counteract the activity of IL-6
by inhibiting the IL-6 receptor, which helps to support the differentiation of a stable Foxp3+ RORγt+ lineage with regulatory functions
(Mucida et al., 2007).
The concurrence of factors that exert protective actions on the
epithelial barrier, together with specific and diversified Treg cells, induced by peptide OIT and, possibly, by the gut microbiota, may aid to
promote intestinal homeostasis in a reciprocal positive manner. While
the mutual contribution of peptides and bacteria to driving intestinal
tolerance deserves further investigation, this study provides evidence
for the role of food peptides in the resolution of food allergy through the
enhancement of vitamin A metabolism and the development of cells
bearing the transcription factor RORγt.
Acknowledgements
This study was funded by the projects AGL2017-88964-R and
AGL2016-75951-R, and L P-R is recipient of a FPU grant (Ministerio de
Ciencia, Innovación y Universidades).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jff.2018.11.012.
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5. Ethics statement
All protocols involving animals followed the European legislation
(Directive 2010/63/EU) and were approved by Comunidad de Madrid
(Ref PROEX 089/15).
Conflict of interest
The authors declare no conflict of interest.
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