IL-2-Agonist-Induced IFN-γ Exacerbates Systemic Anaphylaxis in Food Allergen-Sensitized Mice

Abstract

Food allergies are common, costly and potentially life-threatening disorders. They are driven by Th2, but inhibited by Th1 reactions. There is also evidence indicating that IL-2 agonist treatment inhibits allergic sensitization through expansion of regulatory T cells. Here, we tested the impact of an IL-2 agonist in a novel model for food allergy to hen´s egg in mice sensitized without artificial adjuvants. Prophylactic IL-2 agonist treatment expanded Treg populations and inhibited allergen-specific sensitization. However, IL-2 agonist treatment of already sensitized mice increased mast cell responses and allergic anaphylaxis upon allergen re-challenge. These effects depended on allergen-specific IgE and were mediated through IFN-γ, as shown by IgE transfer and blockade of IFN-γ with monoclonal antibodies. These results suggest that although shifting the allergic reaction toward a Treg/Th1 response inhibits allergic sensitization, the prototypic Th1 cytokine IFN-γ promotes mast cell activation and allergen-induced anaphylaxis in individuals that are already IgE-sensitized. Hence, while a Th1 response can prevent the development of food allergy, IFN-γ has the ability to exacerbate already established food allergy.

Full text

IL-2-Agonist-Induced IFN-g
Exacerbates Systemic Anaphylaxis
in Food Allergen-Sensitized Mice
Christopher W.M. Link
1†
, Christina N. Rau
1†
, Christopher C. Udoye
1
, Mohab Ragab
2
,
Rabia Ü. Korkmaz
1
, Sara Comdühr
1
, Ann-Katrin Clauder
1
, Timo Lindemann
1
,
Britta Frehse
1
, Katharina Hofmann
1
, Larissa N. Almeida
1
, Yves Laumonnier
1
,
Asmaa El Beidaq
1
, Fred D. Finkelman
3‡
and Rudolf A. Manz
1
*
‡
1
Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany,
2
Institute of Nutritional Medicine,
University of Lübeck, Lübeck, Germany,
3
Division of Allergy, Immunology and Rheumatology, Department of Internal
Medicine, University of Cincinnati College of Medicine and the Division of Immunobiology, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH, United States
Food allergies are common, costly and potentially life-threatening disorders. They are
driven by Th2, but inhibited by Th1 reactions. There is also evidence indicating that IL-2
agonist treatment inhibits allergic sensitization through expansion of regulatory T cells.
Here, we tested the impact of an IL-2 agonist in a novel model for food allergy to hen´s egg
in mice sensitized without artificial adjuvants. Prophylactic IL-2 agonist treatment
expanded Treg populations and inhibited allergen-specific sensitization. However, IL-2
agonist treatment of already sensitized mice increased mast cell responses and allergic
anaphylaxis upon allergen re-challenge. These effects depended on allergen-specific IgE
and were mediated through IFN-g, as shown by IgE transfer and blockade of IFN-gwith
monoclonal antibodies. These results suggest that although shifting the allergic reaction
toward a Treg/Th1 response inhibits allergic sensitization, the prototypic Th1 cytokine
IFN-gpromotes mast cell activation and allergen-induced anaphylaxis in individuals that
are already IgE-sensitized. Hence, while a Th1 response can prevent the development of
food allergy, IFN-ghas the ability to exacerbate already established food allergy.
Keywords: food allergy, IL-2, IFN-g, anaphylaxis, murine model
INTRODUCTION
The Th2 cytokine IL-4 promotes class switching to IgE antibodies, which bind to the high affinity
IgE receptor FceRI on mast cells (1–3). Crosslinking of FceRI-bound IgE by antigen/allergen can
induce type-1 hypersensitivity reactions, including those responsible for food allergies (4,5). Th1
reactions and their prototypic cytokine IFN-gcan counterbalance Th2 responses, which reduces the
production of IL-4 and IgE and thereby inhibits allergic sensitization (6,7). These observations
suggested that restoring the Th1/Th2 balance by increasing Th1 responses could inhibit IgE-
mediated allergy (8). This concept is generally well accepted, although it is clear that additional T
cell subsets, such as regulatory T cells (Tregs), also limit allergic reactions (9).
Allergy to chicken (Gallus gallus) egg is the second most prevalent food allergy in infants and
children, after cow’s milk allergy (10). A recent meta-analysis of European studies suggests an
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967721
Edited by:
Eric Cox,
Ghent University, Belgium
Reviewed by:
Hans Oettgen,
Boston Children’s Hospital and
Harvard Medical School, United States
Franz Puttur,
Imperial College London,
United Kingdom
*Correspondence:
Rudolf A. Manz
Rudolf.Manz@uksh.de
†
These authors have contributed
equally to this work and share first
authorship
‡
These authors have contributed
equally to this work and share senior
authorship
Specialty section:
This article was submitted to
Mucosal Immunity,
a section of the journal
Frontiers in Immunology
Received: 20 August 2020
Accepted: 12 November 2020
Published: 11 December 2020
Citation:
Link CWM, Rau CN, Udoye CC,
Ragab M, Korkmaz RÜ, Comdühr S,
Clauder A-K, Lindemann T, Frehse B,
Hofmann K, Almeida LN,
Laumonnier Y, Beidaq AE,
Finkelman FD and Manz RA (2020) IL-
2-Agonist-Induced IFN-gExacerbates
Systemic Anaphylaxis in Food
Allergen-Sensitized Mice.
Front. Immunol. 11:596772.
doi: 10.3389/fimmu.2020.596772
ORIGINAL RESEARCH
published: 11 December 2020
doi: 10.3389/fimmu.2020.596772

overall prevalence of 1.6% in children aged 2.5 years (11).
Chicken egg white (EW) contains several allergenic proteins.
The dominant allergenic protein in EW is the trypsin inhibitor
ovomucoid, which accounts for approximately 11% of EW
protein, while ovalbumin (OVA), the most abundant egg
allergen, accounts for 55% of EW protein. Two putative
allergens have been found in egg yolk (EY), chicken serum
albumin a-livetin and yolk glycoprotein 42 (12,13). a-livetin
has also been identified as the major inhalant allergen in bird-to-
egg syndrome (14,15), which links respiratory hypersensitivity
to bird antigens to egg allergy (16). Exposure to pet birds,
poultry, etc. can lead to respiratory allergies, including asthma,
by sensitizing people to airborne avian allergens (17–21). Blood,
feathers and droppings can be sources of these aeroallergens (22,
23), including serum albumin a-livetin. Reciprocal cross-
inhibition of IgE-binding between extracts of bird feathers and
egg yolk has been observed for patients diagnosed with bird to
egg syndrome (24). Thus, although inhalation of avian
airborne allergens first leads to respiratory hypersensitivity,
subsequent ingestion of cross-reacting serum albumin in egg
yolk can provoke gastro-intestinal symptoms, including diarrhea
and vomiting.
T regulatory cells (Tregs) are important for maintaining oral
tolerance and preventing food allergy (25). These cells can
suppress the production of type 2 cytokines, B cell Ig isotype
switching to IgE and the effector functions of mast cells and
basophils (26). Tregs constitutively express high levels of CD25, a
required component of the high-affinity receptor for IL-2, which
maintains Treg homeostasis and survival in response to IL 2 (27–
29). In the absence of CD25, CD122, and CD132 form a lower
affinity receptor for this cytokine, which is predominantly
expressed by effector CD8+ T cells and natural killer (NK)
cells (30,31). Accordingly, application of low doses of IL-2 is
believed to selectively expand Treg populations, and has
demonstrated therapeutic potential against some autoimmune
disorders in clinical trials (32–35). Interestingly, it was reported
that this treatment inhibits allergic symptoms, but does not
impair IgE production in a murine model of food allergy to
OVA (36).
IL-2 can be complexed with a mAb that blocks its binding to
the low affinity form of the IL-2R to increase its selectivity for
cells, such as Tregs, that express the high affinity IL-2R. In mice,
that can be achieved with the anti-CD122 mAb JES6.
Complexing IL-2 with JES6 also increases its in vivo half-life
and activity (37). Accordingly, IL-2/JES6 is a potent IL-2 agonist
and treatment with IL-2/JES6 can suppress some inflammatory
diseases (38–41). IL-2/JES6 can expand both “natural Tregs”and
“peripheral Tregs”, two distinct Treg subpopulations that are
respectively generated in the thymus without exposure to foreign
antigens, or formed after antigen contact in the periphery,
respectively (42).
In the current study, we physiologically sensitize mice to egg
allergens by intra-tracheal (i.t.) inoculation with EW and EY
plasma (EYP, the liquid, lipid-containing fraction of EY)
without artificial adjuvant. Upon subsequent oral challenge with
the same allergens, the sensitized mice developed diarrhea and
anaphylaxis, which manifests as hypothermia. Using this model
we found that IL-2/JES6 induces an IFN-gresponse; while this
response inhibits Th2 cytokine and IgE production during the
sensitization phase, it reduces the threshold for IgE-mediated mast
cell activation in already sensitized mice.
MATERIALS AND METHODS
Mice
Female Balb/c mice were purchased from Charles River
Laboratories (Sulzfeld, Germany) and maintained under
pathogen-free housing conditions. Animal studies were
performed at the animal facilities of Cincinnati Children’s
Hospital Medical Center and the University of Lübeck, with
approval from the respective authorities.
IL 2/JES6-complex treatment: IL 2/JES6-complex was
prepared by mixing multiples of 1 µg recombinant mouse IL 2
(Immunotools) with 5 µg anti-IL 2 mAb (clone JES6-1A12;
purified from culture supernatants of the hybridoma),
dissolved in 200 µl sterile Dulbecco’s phosphate buffered saline
(DPBS). The solution was incubated for 30 min at 37°C in a
CO2-incubator. IL 2/JES6-complexes were administered i.p. as
described (42).
IFN-g-neutralizing antibody treatment: 100 µg anti-IFN-g
mAb (clone: XMG1.2; kindly donated by Katrin Luger, DRFZ
Berlin.) in 100 µl PBS was intra-peritoneal administered (i.p.) to
allergic mice either one, or three times on three consecutive days,
together with IL-2/JES6.
Experimental Food Allergy to Hen’s Egg
Eggs from the local grocery were swabbed for 5 min with 70%
ethanol and subsequently irradiated for 2 min with UV light. EW
and EY were separated in autoclaved beakers. The EW was
transferred to dialysis tubing (MWCO 6.000–8.000 Da), then
dialyzed against distilled water for 48 h at 4°C, lyophilized, and
stored at 20°C. EY was diluted 1:3 in sterile DPBS and then
centrifuged for 10 min at 13,000 x g, 4°C, after which the
supernatant, EYP, was collected and stored at 20°C.
Mice were anesthetized by i.p. injection of 200 mlanesthetics(5
mg/ml Ketanest S, 1.5 mg/ml Rompun in DPBS), restrained and
sensitized by i.t. application of 40 µl EYP containing 50 µg EW.
The procedure was repeated according to the sensitization
schedules. For antigen (Ag) challenges, lyophilized EW was
dissolved in sterile DPBS to a concentration of 500 mg/ml, then
mixed with an equal volume of EYP. To assess diarrhea
development, EW plus EYP was supplemented with food dye.
Mice were intra-gastrically challenged with 300 µl of this mixture.
Body temperature was measured by rectal thermometry
(Physitemp). IgE-Transfer Model of Passive Systemic Anaphylaxis
Mice were i.v. injected with 10 µg of IgE-anti-TNP mAb (clone
IgEL2a; purified from hybridoma culture supernatants) in 200 µl
sterileDPBS.Toinducesystemicanaphylaxis24haftersensitization,
mice were challenged by gastric lavage with 20 mg of TNP-BSA.
Body temperature was measured by rectal thermometry.
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967722

ELISAs
To determine anti-OVA IgE and IgG1 levels, white Costar®96-
well plates were coated with 10 µg/ml OVA in Tris/saline buffer
(pH 7.2). Briefly, wells were washed and subsequently blocked
with 2% skim milk solution. Standards (IgE (Serotec) and IgG1
(Sigma-Aldrich)) were serially diluted 1:2, starting at 200 ng/ml
or 500 ng/ml, respectively. After incubation with standards and
diluted samples, wells were repeatedly washed and subsequently
incubated with biotinylated anti-mouse IgE (BD) or anti-mouse
IgG1 (Southern Biotech). Wells were then washed and incubated
with Streptavidin-HRP (Thermo Scientific). Following further
washing, substrate (Thermo Scientific) was added to the wells
and responses were immediately measured with a Luminometer
(FlUOstar Omega, BMG Labtech). For quantification of serum
MMCP-1, serum was obtained 4 h after oral gavage (o.g.)
challenge. MMCP-1 concentrations were determined by ELISA
following the manufacturer’s instructions (eBiosciences).
In Vivo Cytokine Capture Assay
IVCCA was performed as described (43). Briefly, mice were i.v.
injected with 10 µg of biotinylated anti-mouse IL-4 (clone
BVD4-1D11) either alone or in combination with 10 µg of
biotinylated anti-mouse IL-13 (clone SZ45-54D1) 4 h before
o.g. challenge. Blood was sampled 4 h after o.g. challenge. Serum
IL 4 and IL 13 levels were measured by luminescence ELISA.
ILC Isolation
Mice were sacrificed and the small intestine was extracted, flushed
with ice cold 1×Hank’s balanced salt solution (without Ca
2+
and
Mg
2+
; HBSS (w/o)) and patches were removed. Lamina propria
lymphocytes were isolated from the entire small intestine using the
isolation kit from Miltenyi Biotech (#130-097-410) according to
the manufacturer’s manual with slight modifications.
Flow Cytometry
Single-cell suspensions were prepared from spleen, mesenteric
LN and mediastinal LN. Subsequently, they were filtered through
a 70-µm cell strainer, washed, and resuspended in PBS/BSA
containing anti-FcgRIIB/RIII mAb (clone 2.4G2) for 5 min.
eF450-labeled anti-CD4 (GK1.5; eBioscience) and Brilliant
Violet 421-labeled anti-CD3 (145-2C11; Biolegend) mAbs were
used for surface staining.
For analysis of ILC, lamina propria lymphocytes were isolated
as described above and stained for: CD3, (clone 17A2; Biolegend)
B220 (clone RA3-6B2; Biolegend), CD11b (clone M1/70;
Biolegend), Gr-1 (clone RB6-8C5; Biolegend), Ter-119 (clone
TER-119; Biolegend), CD49b (clone DX5; Biolegend), CD326
(clone G8.8; Biolegend), CD25 (clone 3C7; Biolegend), CD127
(clone A7R34; Biolegend) Sca-1 (clone D7; Biolegend) and
CD117/c-kit (2B8; BD).
For analysis of intracellular cytokine expression, cells were re-
stimulated by PMA/ionomycin and subsequently protein
secretion was inhibited by Brefeldin A. Then cells were stained
for lineage markers as described above, washed once with PBS,
fixed with fixation solution (BioLegend) according to the
manufacturer’s protocol and permeabilized with the FoxP3
Perm buffer (BioLegend), for 15 min at RT. Subsequently, cells
were washed with ice-cold PBS and intra cellular cytokines IL-10
and IFN-gwere stained by incubation with BV605 labeled anti-
IL-10 mAb (clone: JES5-16E3, Biolegend) and APC conjugated
anti-IFN-gmAb (XMG1.2, Biolegend) for 60 min at RT.
Chloroacetate Esterase (CAE) Staining
Paraffin slides of formaldehyde-fixedjejunumandduodenumwere
de-paraffinized in a Rotihistol (Roth) bath (3 changes, 10 min. per
change) followed by ethanol (Chemsolut) baths of decreasing
concentrations (100%, 95%, 70% V/V). De-paraffinized slides
were rinsed with phosphate buffer (0.1M, pH 7.6). Washed slides
were stained with freshly prepared solutions. First, a new fuchsin
solution (40 mg/ml in 2N HCl; Sigma) was mixed with 4% sodium
nitrite (Sigma). Meanwhile, naphtol AS-D chloroacetate (2 mg/ml
in N,N dimethylformamid; Sigma both) was mixed with phosphate
buffer (0.1M, pH 7.6). The new fuchsin-sodium nitrite solution was
combined with the naphtol AS-D chloroacetate solution, added
dropwise to the slides and incubated for 25 min. The stained slides
were washed with distilled water, counterstained with hematoxylin
gill´s no. 2 (Sigma) and washed with warm tap water. Slides were
dehydrated in an inverse order from the de-paraffination and
sealed under coverslips with Vectamount (Vector).
Statistical Analysis
Data analysis was performed using GraphPad PRISM 6 software,
statistical tests are indicated in individual figure legends. A
p-value <0.05 was considered significant.
RESULTS
Prophylactic IL-2/JES6-Treatment
Prevents IgE Production and
Sensitization to Hen’s Egg
Hen’s egg allergy was induced by i.t. sensitization and oral challenge
with EW plus EYP. Before sensitization, Treg populations were
expanded by three injections of the IL-2 agonist IL 2/JES6 (Figure
1A). Treatment with IL 2/JES6 increased percentages and numbers
of CD3+ CD4+ FoxP3+ Tregs in spleen, blood and mesenteric
lymph nodes approximately 1.5-fold (Figure 1B;Figure S1). After
sensitization, blood samples were collected to evaluate immune
sensitization to hen’s egg. Total IgE and OVA-specificserumIgE
and IgG1 levels were all approximately 5-fold reduced in mice
treated with IL 2/JES6 (Figure 1C). Control mice that were
sensitized to and subsequently challenged with hen’segg
developed anaphylaxis (manifestedashypothermia)asearlyasthe
sixth oral challenge with EW plus EYP; hypothermia plateaued at a
decrease of ~3.0°C by the eighth challenge (Figure 1D). 83% of these
mice also developed allergic diarrhea subsequent to antigen
challenge six. In contrast, mice treated with IL 2/JES6 were
completely protected from allergic reactions, with no hypothermia
and development of diarrhea in only 6.6% (Figure 1D).
In order to investigate possible effects of IL-2/JES6 treatment
on sensitivity to histamine, mice were prophylactic treated with
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967723

A
B
D
C
FIGURE 1 | Prophylactic IL-2/JES6 treatment. (A) Experimental scheme. Mice were treated with IL 2/JES6 or saline (up-arrows) and sensitized by i.t. inoculations
with egg white (EW) plus egg yolk plasma (EYP), as indicated (down-arrows). Later, mice were repeatedly challenged with EW plus EYP. (B) CD3+ CD4+ FoxP3+
Treg populations in spleen after the 12th challenge, analyzed by flow cytometry. (C) Total serum IgE, ovalbumin (OVA)-specific IgE and IgG1 after six i.t.
sensitizations, as assessed by ELISA. (D) Rectal temperature curve (left) and diarrhea development (right) after oral antigen challenges six to eight. Data shown from
one of two independent experiments (n = 14 15). Data depicted represent mean ± SEM. ***p < 0.001, ****p < 0.0001 (Student’s t-test).
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967724

IL-2/JES6, subsequently challenged with allergen and finally
injected with histamine. Compared to a control group treated
with saline, IL-2/JES6 treatment had no effect or slightly
increased histamine-induced hypothermia (Figure S2). Hence,
preventive treatment with the IL-2 agonist IL-2/JES6 blocked
anaphylaxis in our model, most likely by expanding Treg
populations and decreasing allergen-specific IgE production,
rather than by inhibiting the response to histamine. These
findings are in accordance with previous findings obtained in
murine food allergy to milk proteins (44).
After Sensitization, IL-2/JES6-Treatment
Increases Mast Cell Responsiveness and
Exacerbates Acute Allergic Symptoms
In order to test IL 2/JES6 in a therapeutic rather than a
prophylactic setting, the IL-2 agonist was applied to mice that
were already allergic. Repeated challenges with EW plus EYP
induced an allergic response in sensitized mice, as manifested by
hypothermia and diarrhea (Figure 2A). Starting one day after the
11th challenge, allergic mice were treated with IL 2/JES6 on three
consecutive days. Four hours after the last IL 2/JES6 treatment,
mice received a single oral allergen challenge. Strikingly, all IL 2/
JES6-treated mice challenged with allergen developed severe
hypothermia and lethal anaphylaxis while the body temperature
of control mice decreased much less, comparable to previous
challenges (Figure 2B). IL 2/JES6 treatment did not alter the
frequencies of innate lymphoid cells (ILC) in the small intestine of
allergic mice (Figure S3), indicating that IL 2/JES6 may have no
immediate effect on intestinal IL2C.
Mice challenged 72 h after IL 2/JES6 treatment still developed
increased systemic anaphylaxis (Figure 2C), although less severe
than mice challenged 4 h after IL 2/JES6 treatment. As indicated
by increased MMCP-1 levels, IL 2/JES6 treatment led to an
exacerbated mast cell response, while OVA-specific IgE levels
were not affected (Figure 2D). In order to determine if allergen-
sensitization alone is sufficient to induce susceptibility to IL-2/
JES6-mediated exacerbation of anaphylaxis, mice were treated
with IL 2/JES6 directly after allergen sensitization but before the
first oral allergen challenge (Figure 3A). Although saline-treated
mice developed neither hypothermia nor diarrhea to the first oral
challenge, sensitized and IL 2/JES6-treated mice developed an
approximately 5°C drop (Figure 3B). Consistently, compared to
controls, IL 2/JES6-treated mice showed substantially increased
MMCP 1, IL-4 and IL-13 levels in serum (Figure 3C).
Together, these experiments demonstrate that IL-2/JES6
administration that is first administered after mice have been
sensitized does not affect allergen-specific IgE, but exacerbates
anaphylaxis and increases the allergen-reactivity of IgE-sensitive
effector cells, most likely mast cells. To investigate the role of Ag-
specific IgE further, we used an IgE transfer model of systemic
anaphylaxis that depends solely on mast cell activation, but
circumvents the induction of an allergen-specific B and T cell
response (3,45). Mice were treated with three daily doses of IL-2/
JES6. Two days after the last IL-2/JES6 dose they were passively
sensitized with a single dose of monoclonal murine IgE-anti-
trinitrophenyl (TNP) and 24 h later challenged with TNP-BSA
(Figure 4A). IL-2/JES6 treatment increased the anaphylactic and
MMCP-1 responses to allergen-challenge in mice that had
received IgE-anti-TNP (Figures 4B, C). Thus, the IL-2/JES6-
induced increase in mast cell degranulation and anaphylaxis
severity is at least partially independent of increased Ag-specific
IgE levels.
A
B
D
C
FIGURE 2 | Therapeutic IL-2/JES6 treatment. Sensitized and repeatedly
challenged mice were treated with IL-2/JES6 either four or 72 h before the
final allergen challenge. (A) Hypothermia and diarrhea following allergen
challenges, before IL-2/JES6 administration. (B) Rectal temperature curve
(left) and maximal temperature drop (right) of mice treated with IL-2/JES6 4 h
before allergen challenge. (C) Rectal temperature curve (left) and maximal
temperature drop (right) of mice treated with IL-2/JES6 at 72 h before
allergen challenge. (D) Total IgE, ovalbumin (OVA)-specific IgE and IgG1 and
MMCP-1 in serum measured by ELISA (after challenge 13, 72 h after the last
IL-2/JES6 treatment). Data depicted represent mean ± SEM (A: n = 35,
(B–D) n = 7 8). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student’s t-test).
Link et al. IFN-gExacerbates Food Allergy
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Collectively, these data indicate that the IL-2 agonist increases
the allergen-responsiveness of mast cells in the presence of IgE,
which under pathophysiological conditions is the case in already
sensitized individuals.
IFN-gMediates Exacerbation of Acute
Anaphylaxis by IL-2/JES6
To determine how IL-2/JES6 increases IgE-mediated mast cell
responses and exacerbates anaphylaxis, we investigated the
effects of IL-2/JES6 treatment on the size of mast cell
populations in the gut, early events in mast cell activation, and
cytokine responses by T helper cells.
Allergic mice repeatedly treated with IL-2/JES6 did not have
increased mast cell populations in either the mucosa or
connective tissues of the jejunum and the duodenum (Figure
5); thus, IL-2 agonist neither promotes mast cell proliferation nor
increases mast recruitment to the gut.
In order to test whether IL-2/JES6 has an immediate effect on
mast cell activation, mice were sensitized to TNP by injection of
IgE-anti-TNP and received a single injection of the IL-2 agonist
20 h later. Another 4 h later, the mice were challenged with TNP-
BSA. After this single, short treatment, IL-2/JES6 did not promote
anaphylaxis (Figure S4), suggesting that more prolonged treatment
with the IL-2 agonist is required to affect mast cell responsiveness.
A
B
C
FIGURE 3 | IL-2/JES6 treatment after allergen sensitization. Mice were treated with IL-2/JES6 after sensitization and before the first challenge. (A) Treatment
regimen. (B) Rectal temperature curve (left) and maximal temperature drop (right) after antigen challenge. (C) Serum mMCP-1 as assessed by ELISA. IL-4 and IL-13
were measured by IVCCA. Data shown represent mean ± SEM (n = 6). ***p < 0.001, ****p < 0.0001 (Student’s t-test).
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967726

While IL-2/JES6 neither had immediate effects on mast cells,
nor led to the expansion of mast cell populations, repeated
applications of the IL-2 agonist increased the frequencies of
IFN-g+ cells within the CD4+ population, but had no impact on
the frequencies of IL-4+/CD4+, IL-10+/CD4+, and IL-17A+/
CD4+ cells (Figure 6;Figure S5).
Of note, although IFN-gis the prototypic Th1 cytokine and is
known to counteract type-1 hypersensitivity, it has been
previously reported to promote mast cell effector functions and
contribute to disease pathology in a mouse model of chronic
asthma (46). Therefore, we tested whether the IL-2/JES6-induced
increase in IFN-gproduction could be responsible for the
increased mast cell response and exacerbated anaphylaxis
observed in allergic mice. Mice were sensitized to hen’s egg and
challenged repeatedly with EW plus EYP until they developed
considerable titers of allergen-specific IgE but did not yet have
diarrhea or hypothermia following allergen challenge.
Afterwards, the mice were divided into various groups showing
comparable IgE titers. These groups were treated on three
consecutive days with either IL-2/JES6 or saline as a control,
and received one or three injections of anti- IFN-gblocking or
control mAb. The mice were challenged 4 h after the last injection
of anti-IFN-gblocking antibodies. As before, mice not treated
with the IL-2 agonist did not develop detectable temperature
drops. In contrast, all mice treated with the IL-2 agonist but
without IFN-gblockade consistently showed temperature drops
of approximately 5°C (Figures 7A, B). IFN-gblockade
completely inhibited the IL-2/JES6-induced temperature drops.
In addition, mice treated with IL 2/JES6 had increased serum
levels of mast cell-derived proteases that were also reversed by
anti-IFN-gblocking antibodies (Figure 7C). The IFN-g
dependence of IL-2/JES6 exacerbation of anaphylaxis and mast
cell degranulation probably reflects a direct effect of IFN-gon
mast cells, because the addition of IFN-gto cell cultures of bone
marrow-derived mast cells generated from wild-type mice, but
not from IFN-greceptor-deficient mice, has been shown to
enhance mast cell responses in vitro (46).
Collectively, these results show that treating allergic mice
with the IL-2 agonist IL 2/JES6 increases the IFN-gresponse;
which enhances IgE-mediated mast cell degranulation and
consequently exacerbates allergic anaphylaxis.
DISCUSSION
Here, we established a novel model of food allergy to whole egg
that involves sensitization through a biologically relevant route
(the airways) without a requirement for an exogenous adjuvant.
Using this model, we confirmed that IL-2 agonist treatment prior
initial exposure to allergen, inhibits allergic sensitization and
prevents allergen challenge-induced anaphylaxis. However, we
also found that IL-2 agonist treatment drastically intensifies IgE-
dependent mast cell responses and allergic anaphylaxis when
administered to already sensitized mice and that this mechanism,
surprisingly, depends on IFN-g.
Designing experimental models of food al lergy that resemble the
pathophysiology of the human disease provides a challenge (47).
A
B
C
FIGURE 4 | IL-2/JES6 treatment enhances mast cell activation and aggravates IgE-transfer-mediated systemic anaphylaxis. Mice were treated with IL-2/JES6 or
saline, passively sensitized to trinitrophenyl (TNP) by transfer of monoclonal anti-TNP IgE and subsequently challenged with TNP-BSA. (A) Treatment regimen.
(B) Rectal temperature curve (left) and maximal temperature drop (right) following oral challenge. (C) Serum mMCP-1 as assessed by ELISA. Data represent mean ±
SEM (n = 6–7). Data shown from one of two independent experiments. **p < 0.01 (Student’s t-test); *p < 0.05, ****p < 0.0001 (one-way ANOVA followed by Tukey’s
post hoc test).
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967727

Most murine egg allergy models use model antigens, such as OVA,
which initially are applied together with artificial adjuvants.
Intraperitoneal injection with alum adjuvant is the typical route
for sensitization, although this has been replaced, in some
studies, by epicutaneous sensitization through abraded skin.
Intraperitoneal injection of OVA with alum is a strong, albeit
artificial stimulus that allows the use of commercially available
genetically modified mouse strains that express allergen-specificB
and T cell receptors. However, chicken’s egg contains several
allergens. While OVA is the most abundant EW protein,
ovomucoid is the most allergenic (48). Furthermore, in addition
to several protein allergens, hen’s egg contains high concentrations
of saturated fat in egg yolk that appear to act as a natural adjuvant
for the induction of food allergy, an effect that has been observed
with saturated medium chain triglycerides (47)andU.Samavedam
et al., manuscript in preparation. The combination of multiple
protein allergens with lipid adjuvants in egg is likely to be
important in the pathogenesis of food allergy to hen´s egg. In
addition to mimicking the clinically relevant sensitization of
humans with whole egg, rather than a single egg protein, our
airway sensitization scheme has the advantage of mimicking a
natural route of sensitization of humans to eggs, as established for
patients who have developed the bird-to-egg syndrome (49,50)or
have inhaled powdered egg during its manufacture (17–20).
The most novel aspect of our study, however, is not the
sensitization procedure used, but rather the observation that
A
B
FIGURE 5 | Mast cell populations in the gut. Gut sections from sensitized mice treated with IL-2/JES6 or PBS for three days prior to sensitization. Mast cells were
identified by chloroacetate esterase (CAE) staining. (A) Representative section of duodenum, 40x magnification. (B) All mast cells, connective tissue mast cells and
mucosal mast cells in jejunum (upper row) and duodenum (lower row) are shown, as indicated. Plots show mast cells per field of view (MC/field). Upper row:
jejunum, lower row: duodenum, as indicated. Each dot represents the average mast cell counts from one individual mouse, two to six slices were counted per
mouse (n = 5; Mann-Whitney U test). ns, not significant.
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967728

IL-2 exacerbates disease in sensitized individuals by inducing an
IFN-gresponse. Although the Th2 cytokine IL-4 is well known to
play a critical role for the induction of immunoglobulin class
switch to IgE and the development of food allergy (51–54), the
prototypic Th1 cytokine IFN-gcan inhibit allergy by suppressing
IL-4-induced IgE synthesis. Accordingly, shifting the immune
response toward the Th1 phenotype can have beneficial effects
on IgE-mediated allergic diseases (7,55,56).
In contrast, despite its protective role during the initiation of
IgE-mediated allergies, IFN-ghas been shown to contribute to
the pathogenesis of allergic asthma, particularly in patients with
severe disease and during its chronic phase (46,57–59). The
development of full blown disease in one murine model of
chronic asthma has been shown to depend on mast cell
expression of IFN-greceptor-1 (46). IFN-gmediated signals
seem to promote airway inflammation in this model in
multiple ways, including increasing IgE-dependent mast cell
histamine production.
Our data now demonstrate that IFN-gcan also exacerbate
established food allergy. This effect similarly depends on
A
B
FIGURE 6 | Cytokine expression by CD4+ T cells. After challenge 15, allergic mice were therapeutically inoculated for three days with IL-2/JES6 and analyzed 4 h
after the last IL-2/JES6 injection. Cells from mesenteric LNs were stained for lineage markers and intracellular cytokine expression and analyzed by flow cytometry.
(A) Gating. (B) Percentage of IFN-g- and IL-10-expressing CD4+ cells in IL-2/JES6 (circles) and PBS treated controls (squares). Data represent mean ± SEM (n = 10
to 23). **p < 0.01 (two-way ANOVA with Bonferroni past post hoc test).
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5967729

allergen-specific IgE, appears to involve enhanced mast cell
mediator (protease) production of mast cell proteases rather
than an increase in mast cell number (46), takes > 1 day to
develop and dissipates over a few days in the absence of elevated
IFN-glevels. Treatment with the IL-2 agonist IL 2/JES6 led to
increased IFN-gproduction by CD4 T cells, and IFN-gwas
responsible for increased anaphylaxis, most likely through
stimulation of mast cells. Because we show that the IL-2
agonist effect can occur in 3 days or less and IFN-g-dependent
and IL-2 predominantly induces rapid IFN-gproduction by NK
cells (60), it is likely that IL-2 induction of IFN-gproduction by
these cells is also involved.
A
B
C
FIGURE 7 | Impact of IFN-gblockade. Egg-allergic mice were treated daily on three consecutive days with IL-2/JES6 and challenged with allergen after the last IL-2/
JES6 injection. Some groups additionally received one or three injections of anti-IFN-gblocking antibodies, with all or only the last injection of IL-2/JES6, respectively.
Mice were challenged 4 h after the last IL-2/JES6 injection. (A) Temperature drop curve. Representative data from one of two experiments are shown (B) Changes
of maximal temperature drops before and after anti-IFN-gblockade in individual mice (left, statistics: two way ANOVA with Sidak’s multiple comparison test) and
maximal temperature drops after treatment in the various groups (right, statistics: Kruskal-Wallis Test with Dunn’s multiple comparison test) are shown, as indicated.
Data are pooled from two independent experiments. (C) Blood was drawn 1.5 h after the challenge and serum levels of mMCP-1 and mMCP-7 were measured by
ELISA. Data represent mean ± SEM. Representative data from one of two experiments are shown (statistics: Kuskal-Wallis Test with Dunn’s multiple comparison
test). *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 59677210

In addition to the likelihood that IFN-gdirectly enhances the
mast cell response to FceRI-mediated stimulation (46), it is
possible that IFN-gincreases mast cell Ag presentation, which
promotes mutually stimulatory mast cell-T cell interactions that
could enhance effector molecule production, cytokine
production, and mast cell degranulation by reducing the
threshold for mast cells activation via FcgRI (61,62).
Consistent with this possibility, IFN-ghas been shown to
increase mast cell MHC II expression (63,64). Although IFN-g
can inhibit mast cell functioning and even kill mast cells in the
absence of FceRI crosslinking, FceRI crosslinking reverses this
inhibition and increases mast cell antigen presentation via
MHCII (65,66). This may well apply to our model, in which
mast cells are already loaded with allergen-specific IgE when they
encounter increased levels of IFN-gand in which FceRI
crosslinking is induced by allergen-challenge.
Our observation that IL-2-induced IFN-gcan exacerbate food
allergy may appear to contradict previous observations that
endogenously produced IFN-ginhibits allergy. One explanation for
this apparent difference is that IL-2/JES6 treatment of sensitized mice
increases their IL-4 and IL-13 responses to the sensitizing Ag as
observed in our study, in addition to greatly increasing IL-2 levels,
while the TLR agonists and herbal extract used in previous studies to
increase IFN-gproduction suppress the production of Th2 cytokines
(67–70). Consequently, the presence of IL-2, IL-4 and/or IL-13 may
unmask an enhancing effect of IFN-gon mast cell responses, while
IFN-gin the absence of these cytokines has an inhibitory effect.
Our observation that IFN-gexacerbates established food allergy
has clinical implications. The efficacy of low-dose IL-2 therapy,
which has been shown to restore Treg populations in systemic
lupus erythematosus, is currently being evaluated in clinical trials.
Our findings raise the concern that such therapy could be
problematic in food-allergic patients. The impact of IFN-g
during established food allergy reveals also novel aspects on the
relationship between Th2/IgE-mediated allergies and infections.
IFN-gis produced during the immune response against many
infectious pathogens (71,72). The hygiene hypothesis stipulates
that a low prevalence of infection results in a Th2 bias and
eventually contributes to the development of atopic disorders,
including food allergies. However, epidemiological studies have
failed to demonstrate a clear negative correlation between food
allergy and infection. While some papers report that infection with
Helicobacter pylori and Epstein-Barr virus increases the risk of
developing food allergy (73–76), other studies found no correlation
between Helicobacter pylori infection and food allergy or showed
that this infection is associated with a decreased food allergy risk
(77,78). These inconsistent results are consistent with our
observation that IFN-gcan inhibit food allergy during the
sensitization stage, but exacerbate established food allergy.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by local
Committee on the Ethics of Animal Experiments of the state
Schleswig-Holstein (Ministerium für Landwirtschaft, Umwelt
und ländliche Räume des Landes Schleswig-Holstein). Animal
studies performed in Cincinnati were approved by the Cincinnati
Children’s Hospital Medical Center IACUC (IACUC2014-0041,
expires December 12, 2020).
AUTHOR CONTRIBUTIONS
CL, CR, FF, CU, AB, and RM provided substantial contributions
to the conception of the work. CL, CR, CU, MR, RK, SC,
A-KC, TL, BF, KH, LA and AB performed the experiments.
All authors contributed to the article and approved the
submitted version.
FUNDING
This work was supported by the international research training
group “IRTG 1911.”AB, BF, and RM were supported by the
Excellence Cluster “Inflammation at Interfaces,”and KH was
supported by the graduate program “GRK 1727.”A-KC was
funded by the Clinical Research Unit 303 “Pemphigoid Diseases—
Molecular Pathways and their Therapeutic Potential”(CRU303). FF
was supported by NIH grants R01 AI113162, R01AI145991
and R01AI130103.
ACKNOWLEDGMENTS
The authors thank Karsten Seeger, Institute of Chemistry,
University of Lübeck, Germany, for assistance with lyophilizing
the egg white, and Kathleen Kurwahn for her excellent technical
assistance. We thank Stefanie Derer and Heidi Schichting,
Institute for Nutritional Medicine, University of Lübeck
(UKSH) for assistance with paraffin embedding and cutting of
formalin fixed gut. Also, the authors thank Elke Luger,
University Medicine Berlin, Department of Rheumatology, and
German Arthritis Research Center, Berlin, Germany, for the kind
provision of anti-IFN-gmAb (clone: XMG1.2).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.
596772/full#supplementary-material
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 59677211

REFERENCES
1. Tong P, Wesemann DR. Molecular Mechanisms of IgE Class Switch
Recombination. Curr Top Microbiol Immunol (2015) 388:21–37.
doi: 10.1007/978-3-319-13725-4_2
2. Finkelman FD, Rothenberg ME, Brandt EB, Morris SC, Strait RT. Molecular
mechanisms of anaphylaxis: lessons from studies with murine models.
J Allergy Clin Immunol (2005) 115:449–457; quiz 458. doi: 10.1016/
j.jaci.2004.12.1125
3. Finkelman FD. Anaphylaxis: lessons from mouse models. J Allergy Clin
Immunol (2007) 120:506–515; quiz 516–517. doi: 10.1016/j.jaci.2007.07.033
4. Sicherer SH, Sampson HA. Food allergy: A review and update on
epidemiology, pathogenesis, diagnosis, prevention, and management.
J Allergy Clin Immunol (2018) 141:41–58. doi: 10.1016/j.jaci.2017.11.003
5. Wawrzyniak P, Akdis CA, Finkelman FD, Rothenberg ME. Advances and
highlights in mechanisms of allergic disease in 2015. J Allergy Clin Immunol
(2016) 137:1681–96. doi: 10.1016/j.jaci.2016.02.010
6. Maggi E. The TH1/TH2 paradigm in allergy. Immunotechnol Int J Immunol
Eng (1998) 3:233–44. doi: 10.1016/s1380-2933(97)10005-7
7. Romagnani S. The role of lymphocytes in allergic disease. J Allergy Clin
Immunol (2000) 105:399–408. doi: 10.1067/mai.2000.104575
8. Durham SR, Till SJ. Immunologic changes associated with allergen
immunotherapy. J Allergy Clin Immunol (1998) 102:157–64. doi: 10.1016/
s0091-6749(98)70079-x
9. Sampath V, Nadeau KC. Newly identified T cell subsets in mechanistic studies
of food immunotherapy. J Clin Invest (2019) 129:1431–40. doi: 10.1172/
JCI124605
10. Sicherer SH, Sampson HA. Food allergy. J Allergy Clin Immunol (2010) 125:
S116–125. doi: 10.1016/j.jaci.2009.08.028
11. Nwaru BI, Hickstein L, Panesar SS, Roberts G, Muraro A, Sheikh A.
Prevalence of common food allergies in Europe: a systematic review and
meta-analysis. Allergy (2014) 69:992–1007. doi: 10.1111/all.12423
12. De Silva C, Dhanapala P, Doran T, Tang MLK, Suphioglu C. Molecular and
immunological analysis of hen’s egg yolk allergens with a focus on YGP42
(Gal d 6). Mol Immunol (2016) 71:152–60. doi: 10.1016/j.molimm.
2016.02.005
13. Predki PF, Harford C, Brar P, Sarkar B. Further characterization of the N-
terminal copper(II)- and nickel(II)-binding motif of proteins. Studies of metal
binding to chicken serum albumin and the native sequence peptide. Biochem J
(1992) 287( Pt 1):211–5. doi: 10.1042/bj2870211
14. Szepfalusi Z, Ebner C, Pandjaitan R, Orlicek F, Scheiner O, Boltz-Nitulescu G,
et al. Egg yolk alpha-livetin (chicken serum albumin) is a cross-reactive
allergen in the bird-egg syndrome. J Allergy Clin Immunol (1994) 93:932–42.
doi: 10.1016/0091-6749(94)90388-3
15. Quirce S, Marañon F, Umpierrez A, de las Heras M, Fernandez-Caldas E,
Sastre J. Chicken serum albumin (Gal d 5*) is a partially heat-labile inhalant
and food allergen implicated in the bird-egg syndrome. Allergy (2001) 56:754–
62. doi: 10.1034/j.1398-9995.2001.056008754.x
16. Hoffman DR, Guenther DM. Occupational allergy to avian proteins
presenting as allergy to ingestion of egg yolk. J Allergy Clin Immunol (1988)
81:484–8. doi: 10.1016/0091-6749(88)90921-9
17. Bernstein DI, Smith AB, Moller DR, Gallagher JS, Aw TC, London M, et al.
Clinical and immunologic studies among egg-processing workers with
occupational asthma. J Allergy Clin Immunol (1987) 80:791–7. doi: 10.1016/
s0091-6749(87)80267-1
18. Smith AB, Bernstein DI, Aw TC, Gallagher JS, London M, Kopp S, et al.
Occupational asthma from inhaled egg protein. Am J Ind Med (1987) 12:205–
18. doi: 10.1002/ajim.4700120209
19. Smith AB, Bernstein DI, London MA, Gallagher J, Ornella GA, Gelletly SK,
et al. Evaluation of occupational asthma from airborne egg protein exposure
in multiple settings. Chest (1990) 98:398–404. doi: 10.1378/chest.98.2.398
20. Leser C, Hartmann AL, Praml G, Wüthrich B. The “egg-egg”syndrome:
occupational respiratory allergy to airborne egg proteins with consecutive
ingestive egg allergy in the bakery and confectionery industry. J Invest Allergol
Clin Immunol (2001) 11:89–93.
21. Blanco Carmona JG, Juste Picon S, Garces Sotillos M, Rodriguez GastonP.
Occupational asthma in the confectionary industry caused by sensitivity to
egg. Allergy (1992) 47:190–1. doi: 10.1111/j.1398-9995.1992.tb00964.x
22. Mandallaz MM, de Weck AL, Dahinden CA. Bird-egg syndrome. Cross-
reactivity between bird antigens and egg-yolk livetins in IgE-mediated
hypersensitivity. Int Arch Allergy Appl Immunol (1988) 87:143–50. doi:
10.1159/000234664
23. Añibarro Bausela B, Martı

n Esteban M, Martı

nez Alzamora F, Pascual Marcos C,
Ojeda Casas JA. Egg protein sensitization in patients with bird feather allergy.
Allergy (1991) 46:614–8. doi: 10.1111/j.1398-9995.1991.tb00632.x
24. Pabst O, Mowat AM. Oral tolerance to food protein. Mucosal Immunol (2012)
5:232–9. doi: 10.1038/mi.2012.4
25. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of
Treg in immune regulation of allergic diseases. Eur J Immunol (2010)
40:1232–40. doi: 10.1002/eji.200940045
26. Palomares O. The role of regulatory T cells in IgE-mediated food allergy.
J Invest Allergol Clin Immunol (2013) 23:371–382; quiz 2 p preceding 382.
27. Yu A, Zhu L, Altman NH, Malek TR. A low interleukin-2 receptor signaling
threshold supports the development and homeostasis of T regulatory cells.
Immunity (2009) 30:204–17. doi: 10.1016/j.immuni.2008.11.014
28. Malek TR. The biology of interleukin-2. Annu Rev Immunol (2008) 26:453–
79. doi: 10.1146/annurev.immunol.26.021607.090357
29. Yu A, Snowhite I, Vendrame F, Rosenzwajg M, Klatzmann D, Pugliese A, et al.
Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic
mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes.
Diabetes (2015) 64:2172–83. doi: 10.2337/db14-1322
30. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and
activation of the immune system. Nat Rev Immunol (2012) 12:180–90.
doi: 10.1038/nri3156
31. Letourneau S, Krieg C, Pantaleo G, Boyman O. IL-2- and CD25-dependent
immunoregulatory mechanisms in the homeostasis of T-cell subsets. J Allergy
Clin Immunol (2009) 123:758–62. doi: 10.1016/j.jaci.2009.02.011
32. Tahvildari M, Dana R. Low-Dose IL-2 Therapy in Transplantation,
Autoimmunity, and Inflammatory Diseases. J Immunol Baltim Md 1950
(2019) 203:2749–55. doi: 10.4049/jimmunol.1900733
33. Ballesteros-Tato A, Papillion A. Mechanisms of action of low-dose IL-2
restoration therapies in SLE. Curr Opin Immunol (2019) 61:39–45.
doi: 10.1016/j.coi.2019.07.003
34. von Spee-Mayer C, Siegert E, Abdirama D, Rose A, Klaus A, Alexander T,
et al. Low-dose interleukin-2 selectively corrects regulatory T cell defects in
patients with systemic lupus erythematosus. Ann Rheum Dis (2016) 75:1407–
15. doi: 10.1136/annrheumdis-2015-207776
35. Humrich JY, Riemekasten G. Low-dose IL-2 therapy - a complex scenario that
remains to be further explored. Nat Rev Rheumatol (2017) 13:386.
doi: 10.1038/nrrheum.2017.71
36. Bonnet B, Vigneron J, Levacher B, Vazquez T, Pitoiset F, Brimaud F, et al.
Low-Dose IL-2 Induces Regulatory T Cell-Mediated Control of Experimental
Food Allergy. J Immunol Baltim Md 1950 (2016) 197:188–98. doi: 10.4049/
jimmunol.1501271
37. Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J. Selective stimulation
of T cell subsets with antibody-cytokine immune complexes. Science (2006)
311:1924–7. doi: 10.1126/science.1122927
38. Yokoyama Y, Iwasaki T, Kitano S, Satake A, Nomura S, Furukawa T, et al. IL-
2-Anti-IL-2 Monoclonal Antibody Immune Complexes Inhibit Collagen-
Induced Arthritis by Augmenting Regulatory T Cell Functions. J Immunol
Baltim Md 1950 (2018) 201:1899–906. doi: 10.4049/jimmunol.1701502
39. Dinh TN, Kyaw TS, Kanellakis P, To K, Tipping P, Toh B-H, et al. Cytokine
therapy with interleukin-2/anti-interleukin-2 monoclonal antibody
complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates
development and progression of atherosclerosis. Circulation (2012) 126:1256–
66. doi: 10.1161/CIRCULATIONAHA.112.099044
40. Hamano R, Baba T, Sasaki S, Tomaru U, Ishizu A, Kawano M, et al. Ag and
IL-2 immune complexes efficiently expand Ag-specific Treg cells that migrate
in response to chemokines and reduce localized immune responses. Eur J
Immunol (2014) 44:1005–15. doi: 10.1002/eji.201343434
41. Wilson MS, Pesce JT, Ramalingam TR, Thompson RW, Cheever A, Wynn
TA. Suppression of murine allergic airway disease by IL-2:anti-IL-2
monoclonal antibody-induced regulatory T cells. J Immunol Baltim Md
1950 (2008) 181:6942–54. doi: 10.4049/jimmunol.181.10.6942
42. El Beidaq A, Link CWM, Hofmann K, Frehse B, Hartmann K, Bieber K, et al.
In Vivo Expansion of Endogenous Regulatory T Cell Populations Induces
Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 59677212

Long-Term Suppression of Contact Hypersensitivity. J Immunol Baltim Md
1950 (2016) 197:1567–76. doi: 10.4049/jimmunol.1600508
43. Finkelman F, Morris S, Orekhova T, Sehy D. The in vivo cytokine capture
assay for measurement of cytokine production in the mouse. Curr Protoc
Immunol (2003) 54(1):6.28.1–6.28.10. doi: 10.1002/0471142735.im0628s54.
Chapter 6:Unit 6.28.
44. Smaldini PL, Trejo F, Cohen JL, Piaggio E, Docena GH. Systemic IL-2/anti-IL-
2Ab complex combined with sublingual immunotherapy suppresses
experimental food allergy in mice through induction of mucosal regulatory
T cells. Allergy (2018) 73:885–95. doi: 10.1111/all.13402
45. Kucuk ZY, Strait R, Khodoun MV, Mahler A, Hogan S, Finkelman FD.
Induction and suppression of allergic diarrhea and systemic anaphylaxis in a
murine model of food allergy. J Allergy Clin Immunol (2012) 129:1343–8.
doi: 10.1016/j.jaci.2012.03.004
46. Yu M, Eckart MR, Morgan AA, Mukai K, Butte AJ, Tsai M, et al. Identification
of an IFN-g/mast cell axis in a mouse model of chronic asthma. J Clin Invest
(2011) 121:3133–43. doi: 10.1172/JCI43598
47. Kanagaratham C, Sallis BF, Fiebiger E. Experimental Models for Studying
Food Allergy. Cell Mol Gastroenterol Hepatol (2018) 6:356–369.e1.
doi: 10.1016/j.jcmgh.2018.05.010
48. Dhanapala P, De Silva C, Doran T, Suphioglu C. Cracking the egg: An insight
into egg hypersensitivity. Mol Immunol (2015) 66:375–83. doi: 10.1016/
j.molimm.2015.04.016
49. Bartnikas LM, Gurish MF, Burton OT, Leisten S, Janssen E, Oettgen HC, et al.
Epicutaneous sensitization results in IgE-dependent intestinal mast cell
expansion and food-induced anaphylaxis. J Allergy Clin Immunol (2013)
131:451–460.e1–6. doi: 10.1016/j.jaci.2012.11.032
50. Caubet J-C, Wang J. Current understanding of egg allergy. Pediatr Clin North
Am (2011) 58:427–443, xi. doi: 10.1016/j.pcl.2011.02.014
51. Yanagihara Y, Ikizawa K, Kajiwara K, Koshio T, Basaki Y, Akiyama K.
Functional significance of IL-4 receptor on B cells in IL-4-induced human
IgE production. J Allergy Clin Immunol (1995) 96:1145–51. doi: 10.1016/
s0091-6749(95)70199-0
52. Finkelman FD, Katona IM, Urban JF, Holmes J, Ohara J, Tung AS, et al. IL-4
is required to generate and sustain in vivo IgE responses. J Immunol Baltim
Md 1950 (1988) 141:2335–41.
53. Tordesillas L, Berin MC, Sampson HA. Immunology of Food Allergy.
Immunity (2017) 47:32–50. doi: 10.1016/j.immuni.2017.07.004
54. Wu LC, Scheerens H. Targeting IgE production in mice and humans. Curr
Opin Immunol (2014) 31:8–15. doi: 10.1016/j.coi.2014.08.001
55. Lewis DB. Allergy immunotherapy and inhibition of Th2 immune responses:
a sufficient strategy? Curr Opin Immunol (2002) 14:644–51. doi: 10.1016/
s0952-7915(02)00388-6
56. Romagnani S. Immunologic influences on allergy and the TH1/TH2 balance.
J Allergy Clin Immunol (2004) 113:395–400. doi: 10.1016/j.jaci.2003.11.025
57. Branchett WJ, Stölting H, Oliver RA, Walker SA, Puttur F, Gregory LG, et al.
A T cell-myeloid IL-10 axis regulates pathogenic IFN-g-dependent immunity
in a mouse model of type 2-low asthma. J Allergy Clin Immunol (2020)
145:666–678.e9. doi: 10.1016/j.jaci.2019.08.006
58. Raundhal M, Morse C, Khare A, Oriss TB, Milosevic J, Trudeau J, et al. High
IFN-gand low SLPI mark severe asthma in mice and humans. J Clin Invest
(2015) 125:3037–50. doi: 10.1172/JCI80911
59. Poon AH, Eidelman DH, Martin JG, Laprise C, Hamid Q. Pathogenesis of
severe asthma. Clin Exp Allergy J Br Soc Allergy Clin Immunol (2012) 42:625–
37. doi: 10.1111/j.1365-2222.2012.03983.x
60. Thornton S, Kuhn KA, Finkelman FD, Hirsch R. NK cells secrete high levels of
IFN-gamma in response to in vivo administration of IL-2. Eur J Immunol
(2001) 31(11):3355–60. doi: 10.1002/1521-4141(200111)31:11
61. Gaudenzio N, Laurent C, Valitutti S, Espinosa E. Human mast cells drive
memory CD4+ T cells toward an inflammatory IL-22+ phenotype. J Allergy
Clin Immunol (2013) 131:1400–1407.e11. doi: 10.1016/j.jaci.2013.01.029
62. Gaudenzio N, Espagnolle N, Mars LT, Liblau R, Valitutti S, Espinosa E. Cell-
cell cooperation at the T helper cell/mast cell immunological synapse. Blood
(2009) 114:4979–88. doi: 10.1182/blood-2009-02-202648
63. Warbrick EV, Taylor AM, Botchkarev VA, Coleman JW. Rat connective
tissue-type mast cells express MHC class II: up-regulation by
IFN-gamma. Cell Immunol (1995) 163:222–8. doi: 10.1006/
cimm.1995.1120
64. Lotfi-Emran S, Ward BR, Le QT, Pozez AL, Manjili MH, Woodfolk JA, et al.
Human mast cells present antigen to autologous CD4+ T cells. J Allergy Clin
Immunol (2018) 141:311–321.e10. doi: 10.1016/j.jaci.2017.02.048
65. Yanagida M, Fukamachi H, Takei M, Hagiwara T, Uzumaki H, Tokiwa T,
et al. Interferon-gpromotes the survival and FcϵRI-mediated histamine
release in cultured human mast cells. Immunology (1996) 89:547–52.
doi: 10.1046/j.1365-2567.1996.d01-768.x
66. Sellge G, Barkowsky M, Kramer S, Gebhardt T, Sander LE, Lorentz A, et al.
Interferon-gregulates growth and controls Fcgreceptor expression and
activation in human intestinal mast cells. BMC Immunol (2014) 15:27.
doi: 10.1186/1471-2172-15-27
67.KulisM,GorentlaB,BurksAW,ZhongX-P.TypeBCpG
oligodeoxynucleotides induce Th1 responses to peanut antigens:
modulation of sensitization and utility in a truncated immunotherapy
regimen in mice. Mol Nutr Food Res (2013) 57:906–15.doi:10.1002/
mnfr.201200410
68. Srivastava KD, Qu C, Zhang T, Goldfarb J, Sampson HA, Li X-M. Food
Allergy Herbal Formula-2 silences peanut-induced anaphylaxis for a
prolonged posttreatment period via IFN-gamma-producing CD8+ T
cells. J Allergy Clin Immunol (2009) 123:443–51. doi: 10.1016/
j.jaci.2008.12.1107
69. Zhu F-G, Kandimalla ER, Yu D, Agrawal S. Oral administration of a synthetic
agonist of Toll-like receptor 9 potently modulates peanut-induced allergy in
mice. J Allergy Clin Immunol (2007) 120:631–7. doi: 10.1016/j.jaci.2007.05.015
70. San Roman B, Irache JM, Gomez S, Gamazo C, Espuelas S. Co-delivery of
ovalbumin and CpG motifs into microparticles protected sensitized mice from
anaphylaxis. Int Arch Allergy Immunol (2009) 149:111–8. doi: 10.1159/
000189193
71. Infante-Duarte C, Kamradt T. Th1/Th2 balance in infection. Springer Semin
Immunopathol (1999) 21:317–38. doi: 10.1007/bf00812260
72. Szabo SJ, Sullivan BM, Peng SL, Glimcher LH. Molecular mechanisms
regulating Th1 immune responses. Annu Rev Immunol (2003) 21:713–58.
doi: 10.1146/annurev.immunol.21.120601.140942
73. Shabrawy RM, Gharib K. Helicobacter pylori Infection as a Risk Factor in
Patients Suffering from Food Allergy and Urticaria. Egypt J Immunol (2016)
23:67–75.
74. Bartuzi Z, Korenkiewicz J, Romanski B. Correlation between Helicobacter
pylori infection and food allergy in chronic gastritis. Med Sci Monit Int Med J
Exp Clin Res (2000) 6:530–8.
75. Figura N, Perrone A, Gennari C, Orlandini G, Giannace R, Lenzi C, et al. CagA-
positive Helicobacter pylori infection may increase the risk of food allergy
development. JPhysiolPharmacolOffJPolPhysiolSoc(1999) 50:827–31.
76. Pan Y, Nie Z, Zhang Y, Zhang K, Li J, Wang L. Identification of EBV infection
in adults with egg specific food allergy. Virol J (2013) 10:9. doi: 10.1186/1743-
422X-10-9
77. Ma ZF, Majid NA, Yamaoka Y, Lee YY. Food Allergy and Helicobacter pylori
Infection: A Systematic Review. Front Microbiol (2016) 7, 368. doi: 10.3389/
fmicb.2016.00368
78. Konturek PC, Rienecker H, Hahn EG, Raithel M. Helicobacter pylori as a
protective factor against food allergy. Med Sci Monit Int Med J Exp Clin Res
(2008) 14:CR452–458.
Conflict of Interest: The authors declare that the research was conducted in the
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potential conflict of interest.
Copyright © 2020 Link, Rau, Udoye, Ragab, Korkmaz, Comdühr, Clauder,
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Link et al. IFN-gExacerbates Food Allergy
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 59677213