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\begin{document}

\title{IL-25 Promotes Eosinophils Antigen Presenting Function in Allergic
Asthma}



\author[1]{Bo Peng}%
\author[1]{Lin Sun}%
\author[2]{Meng Zhang}%
\author[1]{Huacheng Yan}%
\author[1]{Guochao Shi}%
\author[2]{Zhenwei  Xia}%
\author[1]{Ranran Dai}%
\author[1]{Wei Tang}%
\affil[1]{Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital Department of Respiratory Diseases}%
\affil[2]{Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
\textbf{Background:} Eosinophils act as a secondary antigen-presenting
cell (APC) to stimulate Th cell responses against antigens. IL-25 plays
an important role on eosinophil activation in allergic asthma. The role
of IL-25 on the classic APC functions of dendritic cells has been
elucidated. However, whether IL-25 facilitates eosinophils for antigen
presentation is unknown. \textbf{Objective:} To elucidate IL-25 on
eosinophils antigen presenting function during allergic asthma and its
related mechanism. \textbf{Methods:} Eosinophils from allergic asthma
subjects were cultured with IL-25 and HDM to identify the co-stimulator
molecules expression. Co-cultures of eosinophils and autologous na\selectlanguage{ngerman}ïve
CD4+ T cells in the same culture system were to explore whether
eosinophils had the capacity to promote Th cell differentiation in
response to IL-25 engagement. In asthma mouse model, IL-25
\textsuperscript{-/-} mice were exposed to HDM to investigate IL-25
functions on eosinophils during sensitization phase. The impact of IL-25
on the capacity for eosinophil taking up antigens and costimulatory
molecules expression was evaluated. Mouse bone marrow derived
eosinophils (BmEOS) were co-cultured with naïve CD4+T cells sorted from
spleens under HDM and IL-25 stimulation to identify T cell
differentiation. \textbf{Results:} IL-25 upregulated HLA-DR, PD-L1 and
OX-40L expression on eosinophils from allergic asthma patients. IL-25
and HDM co-sensitized eosinophils promoted Th2 differentiation. In
animal model, IL-25 \textsuperscript{-/-} mice experienced restrained
allergic pulmonary inflammation and reduced eosinophils chemotaxis
during early sensitization phase. In vitro, IL-25 promoted antigen
uptake by eosinophils. During BmEOS and naïve CD4+T cells co-culture,
IL-25 accreted the proportion of CD4+Th2 cells, which was absent in
CD4+T cells culture alone. \textbf{Conclusion:} Our data identify a
novel role of IL-25 in enhancing eosinophils antigen presenting capacity
to induce Th2 priming in the context of allergic inflammation.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{IL-25 Promotes Eosinophils Antigen Presenting Function in
Allergic Asthma}

Running Title:IL-25 facilitates eosinophils APC capacity

Bo Peng\textsuperscript{1,2}*, Lin Sun\textsuperscript{1,2}*, Meng
Zhang\textsuperscript{3}, Huacheng Yan\textsuperscript{1,2}, Guochao
Shi\textsuperscript{1,2}, Zhenwei Xia\textsuperscript{3}+, Ranran
Dai\textsuperscript{1,2}+, Wei Tang\textsuperscript{1,2}+

\begin{enumerate}
\tightlist
\item
  Department of Respiratory and Critical Care Medicine, Ruijin Hospital,
  Shanghai Jiao Tong University School of Medicine,197, Rui Jin Er Road,
  Shanghai, China.
\item
  Shanghai Key Laboratory of Emergency Prevention, Diagnosis and
  Treatment of Respiratory Infectious Diseases, Shanghai, China.
\item
  Department of Pediatrics, Ruijin Hospital, Shanghai Jiao Tong
  University School of Medicine, 197, Rui Jin Er Road, Shanghai, China.
\end{enumerate}

*, + These authors have contributed equally

\textbf{Corresponding author:}

Wei Tang, MD\&PHD, Associate Professor, Department of Respiratory and
Critical Care Medicine, Ruijin Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai, China. Email address:
tina\_tangwei@163.com. Phone number: 86-21-64370045 x680703

Ranran Dai, MD\&PHD, Department of Respiratory and Critical Care
Medicine, Ruijin Hospital, Shanghai Jiaotong University School of
Medicine, Shanghai, China. Email address: drrcyy@msn.com. Phone number:
86-21-64370045 x680805

Zhenwei Xia, MD\&PHD, Department of Pediatrics, Ruijin Hospital,
Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road,
Shanghai 200025, China. Email: xzw10484@rjh.com.cn. Phone number:
+86-21-6437004

\textbf{ACKNOWLEDGEMENT}

We thank the nurses Ping Wang, Jin Cao, Guofang Xu, Yi Tao for their
help in the patient specimen collection and implementation.

\textbf{ABSTRACT}

\textbf{Background:} Eosinophils act as a secondary antigen-presenting
cell (APC) to stimulate Th cell responses against antigens. IL-25 plays
an important role on eosinophil activation in allergic asthma. The role
of IL-25 on the classic APC functions of dendritic cells has been
elucidated. However, whether IL-25 facilitates eosinophils for antigen
presentation is unknown.

\textbf{Objective:} To elucidate IL-25 on eosinophils antigen presenting
function during allergic asthma and its related mechanism.

\textbf{Methods:}Eosinophils from allergic asthma subjects were cultured
with IL-25 and HDM to identify the co-stimulator molecules expression.
Co-cultures of eosinophils and autologous na\selectlanguage{ngerman}ïve CD4+ T cells in the same
culture system were to explore whether eosinophils had the capacity to
promote Th cell differentiation in response to IL-25 engagement. In
asthma mouse model, IL-25\textsuperscript{-/-} mice were exposed to HDM
to investigate IL-25 functions on eosinophils during sensitization
phase. The impact of IL-25 on the capacity for eosinophil taking up
antigens and costimulatory molecules expression was evaluated. Mouse
bone marrow derived eosinophils (BmEOS) were co-cultured with naïve
CD4+T cells sorted from spleens under HDM and IL-25 stimulation to
identify T cell differentiation.

\textbf{Results:} IL-25 upregulated HLA-DR, PD-L1 and OX-40L expression
on eosinophils from allergic asthma patients. IL-25 and HDM
co-sensitized eosinophils promoted Th2 differentiation. In animal model,
IL-25\textsuperscript{-/-} mice experienced restrained allergic
pulmonary inflammation and reduced eosinophils chemotaxis during early
sensitization phase. In vitro, IL-25 promoted antigen uptake by
eosinophils. During BmEOS and naïve CD4+T cells co-culture, IL-25
accreted the proportion of CD4+Th2 cells, which was absent in CD4+T
cells culture alone.

\textbf{Conclusion} \textbf{:} Our data identify a novel role of IL-25
in enhancing eosinophils antigen presenting capacity to induce Th2
priming in the context of allergic inflammation.

\textbf{Keywords}

IL-25, eosinophil, APC, allergic asthma

\textbf{INTRODUCTION}

Allergic asthma is a major and increasingly prevalent chronic
inflammatory disease of the airways which is characterized by airway
infiltration of eosinophils, Th2 cells, basophils and mast cells,
together leading to mucus hypersecretion, airway wall remodeling, and
airway hyperresponsiveness\textsuperscript{1, 2}. The most predominant
and clearly described endotype of asthma is eosinophilic asthma, which
accounts for over 50\% of adults with asthma\textsuperscript{3}.

Eosinophils play pivotal roles in allergic airway inflammation, not only
working in the effector phase to participate in the inflammation, but
also functioning in the early phase to initiate Th2 cell responses,
acting as a non-professional antigen-presenting cell
(APC)\textsuperscript{4, 5}. Accumulating evidence revealed that
eosinophils could express MHCII, migrate to lung and lymph nodes,
process the exogenous antigen and induce proliferative Th2
polarization\textsuperscript{6-8}. What drives the process was not
elucidated yet.

IL-25, also known as IL-17E, has been shown as a key mediator in
allergic asthma, strongly expressed during allergic airway
inflammation\textsuperscript{9}. We previously showed that IL-17RA/RB
expression on eosinophils of allergic asthmatics was significantly
higher than that of atopic non-asthmatics controls and increased
significantly 24 h after receiving allergen inhalation
challenge\textsuperscript{10, 11}. Although the diverse roles of IL-25
on eosinophils including enhancing survival or reducing apoptosis have
been described\textsuperscript{12, 13}, the effect of IL-25 on
eosinophils antigen presentation is unknown. With high expression of
IL-25 receptors on eosinophils in asthma and the capacity of IL-25 to
facilitate classical antigen presentation cell-dendritic cells (DCs) to
initiate Th2 responses\textsuperscript{14-16}, we therefore asked
whether IL-25 possesses the activity to augment eosinophils to initiate
Th2 responses.

Here, we investigated that IL-25 activates the expression of cell
surface molecules on eosinophils and antigen uptake by eosinophils. We
identify that IL-25 could contribute to eosinophil-mediated Th2
polarization both in \emph{ex vivo} murine and human studies. In the
absence of IL-25, eosinophils chemotaxis to lung during sensitization
was hampered, resulting in alleviated airway inflammation and
ameliorated Th2 responses.

\textbf{METHODS}

\textbf{Subjects}

14 subjects with mild-to-moderate allergic asthma were enrolled in this
study. Inclusion criteria included a physician diagnosis of allergic
asthma and serum Der p 1 specific-IgE\textgreater{}0.35kUA/L. Exclusion
criteria included upper respiratory infection within 4 weeks, prednisone
use within 3 months, pregnancy, and other lung diseases or autoimmune
diseases. All patients were taking low or medium dose ICS-LABA as their
daily treatment and were well-controlled. Detailed patient information
is shown in Table S1. The study was approved by the Ethic Committee of
Ruijin Hospital (No.2019-YK061). All participants signed an informed
consent form prior to the study.

\textbf{In vitro eosinophils stimulation assays andco-culture of
eosinophils and autologous naïve CD4+ T cells}

Eosinophils and naïve CD4+ T cells from allergic asthmatics were
separated from peripheral blood by magnetic cell separation system
(MACS) using Eosinophil Isolation Kit (\#130-092-010, Miltenyi) and
Naïve CD4+ T Cell Isolation Kit II (\#130-094-131, Miltenyi) as
previously described\textsuperscript{17}. Purity of isolated cells was
checked by flowcytometry and was always \textgreater{}95\%. Purified
eosinophils were resuspended at 1.0x10\textsuperscript{6} /mL in RPMI
1640(Hyclone) supplemented with 10\% FBS, 1\% penicillin/streptomycin
(Servicebio), and 10ng/ml IL-5 (Peprotech). Eosinophils were stimulated
with HDM (100\selectlanguage{greek}μ\selectlanguage{english}g/ml, Greer Lab, low endotoxin) and/or IL-25(1ng/ml,
Peprotech) at 37\selectlanguage{ngerman}°C in a humidified 5\% CO2 incubator for 18 h, and then
cells were harvested for co-culture or direct flowcytometry analysis.

After 18 h stimulation with HDM and/or IL-25, eosinophils
(1.0x10\textsuperscript{6} /ml) were resuspended with autologous naïve
CD4+ T cells (106 /ml) in 1:1(v:v) RPMI 1640 (Hyclone) with ImunoCult-XF
T Cell Expansion Medium (Stemcell) supplemented with 10ng/ml IL-2
(Peprotech), 10ng/ml IL-5 (Peprotech) and 25\selectlanguage{greek}μ\selectlanguage{english}l/ml ImmunoCult Human
CD3/CD28 T Cell Activator (Stem Cell). Cells were cultured at 37\selectlanguage{ngerman}°C in a
humidified 5\% CO2~incubator for 48h. Individual naïve CD4+ T cells
(1.0x106 /ml) groups were served as control, culturing in the same
condition medium above. 4 h before the end of culture, 1:1000 Monensin
Solution (Biolegend) were added for intracellular cytokines analysis.

The flow cytometry analyses were performed with a Cytoflex LX instrument
(Beckman). Dead cells were excluded by Zombie UV (Biolegend). For
eosinophil surface marker analysis, cells were incubated with Fc
Receptor Blocking Solution (Biolegend) to eliminate Fc receptor-mediated
antibody binding, and then followed with staining of APC/Cy7 anti-human
CD16 antibody(Clone:3G8, BD sciences) , APC anti-human Siglec-8
antibody(Clone:7C9), PE anti-human CD40 antiobody(Clone:HB14),
PE/Cyanine7 anti-human HLA-DR antibody(Clone:L243), Brilliant Violet 421
anti-human CD80 antibody(Clone:L307.4, BD sciences), BB515 anti-human
CD86 antibody (Clone:FUN-1, BD sciences), PE/Cyanine7 anti-human PD-L1
antibody (Clone:29E.2A3), PE anti-human OX-40L antibody(Clone:11C3.1)
for 20 min at 20°C in the dark, then cells were washed, fixed with
Fixation Buffer (Biolgend) and resuspended in Cyto-last Buffer
(Biolegend) until analysis.

To measure cytokines production of T cells after co-culture, following
surface staining of FITC anti-human CD3 antibody (Clone: UCHT1),
PE/Cyanine7 anti-human CD4 antibody (Clone: RPA-T4), cells were fixed by
Fixation Buffer (Biolgend) for 20 min at 20°C avoided from light, and
then washed twice with Intracellular Permeabilization Buffer. Cells were
stained with APC/Fire 750 anti-human IFN-\selectlanguage{greek}γ \selectlanguage{english}antibody (Clone:4S.B3),
Brilliant Violet 421 anti-human IL-4 antibody (Clone:G077F6), PE
anti-human IL-9 antibody (Clone:MH9A4), APC anti-human IL-17A antibody
(Clone:BL168) in remaining permeabilization buffer for 30 min at 20\selectlanguage{ngerman}°C in
the dark, and then washed and resuspended in FACS buffer for analysis.
All antibodies were purchased from Biolegend unless otherwise specified.

\textbf{Mice}

IL-25-deficient mice\selectlanguage{english}(Il25\textsuperscript{-/-}\selectlanguage{english})were obtained from
Tsinghua university, which have been described in the previous
studies\textsuperscript{18, 19}. The genotyping primers were as follows:
forward, 5\selectlanguage{english}'-CTGCTCCAGTCAGCCTCTCT-3\selectlanguage{english}'; reverse1, 5\selectlanguage{english}'AGCAGCTGGGCAAGTGAC-3\selectlanguage{english}';
reverse2,5\selectlanguage{english}' AGGTGGAGAAAGTGCCTGT-3\selectlanguage{english}'. All mice were bred and maintained
under specific pathogen-free conditions in Shanghai Biomodel Organism
Science and Technology Development Co., Ltd. Sex-matched littermate WT
and Il25\textsuperscript{-/-} mice 6 to 8 weeks of age were mainly used
for experiments.

The HDM-induced mouse asthma model was established as described
previously\textsuperscript{20}. Briefly, mice were sensitized
intranasally with 100 \selectlanguage{greek}μ\selectlanguage{english}g house dust mite extracts (HDM) (Greer Labs, low
endotoxin) in 40 \selectlanguage{greek}μ\selectlanguage{english}L PBS on day 0 and challenged intranasally with 10 \selectlanguage{greek}μ\selectlanguage{english}g
HDM on days 7 to 11, harvested on day 14. In some experiments for
investigating the role of eosinophils in the sensitization period, mice
were sensitized with 100 \selectlanguage{greek}μ\selectlanguage{english}g HDM before the first challenge and lungs
were harvested for flow cytometry analysis at indicated times. To study
the effect of IL-25, mice were treated intranasally with 1\selectlanguage{greek}μ\selectlanguage{english}g IL-25(R\&D)
in 40 \selectlanguage{greek}μ\selectlanguage{english}L PBS and lungs were digested in HBSS media containing type 1A
collagenase (Sigma) to obtain the single cell suspension for flow
cytometry analysis 24 hours later. Histopathologic analysis was done to
identify asthma allergic inflammation in mouse models. BALF was
centrifuged and supernatants were frozen at -80 for subsequent cytokine
analysis. Cytokines IL-4, IL-5, IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-13 levels in BALF and serum
IgE levels were measured by ELISA according to the manufacturer's
instructions (BioLegend). All animal studies were approved by the Ruijin
Hospital Animal Ethics Committee.

\textbf{Generation of mouse bone marrow-derived eosinophil (BmEOS) and
DQ-OVA uptake by BmEOS}

The ex vivo culture of BmEOS was adapted from the previously published
protocols with minor modifications\textsuperscript{21, 22}. Briefly,
Bone marrow (BM) was flushed out from the tibia and fibula of C57bl/6J
mice. After erythrocyte lysis, BM cells were seeded at 1\selectlanguage{ngerman}×10\^{}6/ml in
IMDM media supplemented with 20\% fetal bovine serum (Gibco), 1\%
penicillin/streptomycin, 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate
(Gibco), and 50 mM 2-mercaptoethanol (Sigma-Aldrich) in the presence of
100 ng/mL FLT-3L and 100 ng/mL recombinant murine stem cell factor (SCF,
Peprotech), cultured from day 0 to day 4. On day 4 and 8, the media
containing SCF and FLT3-L was replaced with media containing 10 ng/mL
recombinant mouse interleukin-5 (rmIL-5; R\&D Systems) only. Every other
day, from this point forward, media was replaced with fresh media
containing rmIL-5. Cells were evaluated by flow cytometry and performed
from day 10 to 14.

BmEOS were seeded in 48 wells plates and stimulated for 2 days with or
without HDM and IL-25 in complete medium. To assess DQ-OVA uptake by
BmEOS, BmEOS were treated with DQ-OVA (Themofisher, USA) for 6 hours
before being collected, washed and further analyzed using flow
cytometry. As a negative control, cells were cultured in the presence of
DQ-OVA on ice.

\textbf{In vitro Polarization of CD4+ T Cells with BmEOS}

Spleens were removed from C57bl/6J 6\textasciitilde{}8-week-old mice,
mechanically disrupted, treated with RBC lysis buffer and run through 70
\selectlanguage{greek}μ\selectlanguage{english}m cell strainer. Suspensions were resuspended in MACS buffer, labelled
with an antibody cocktail and na\selectlanguage{ngerman}ïve CD4+ T cells were negatively
separated using the mouse Naïve CD4+ T cell Isolation kit (Miltenyi
Biotech), in accordance with manufacturer's instructions. High purity
isolated naïve CD4+ T cells (1*10\^{}6/ml) were co-cultured with the
BmEOS (5*10\^{}5/ml) in supplemented IMDM medium with or without
100ug/ml HDM in the presence or absence of 50 ng/ml IL-25, with IL-2 and
GM-CSF treatment to sustain T cells survival. As controls, CD4+ T cells
alone were also cultured. Cultures were incubated for 96 h at 37°C in a
5\% CO2 humidified atmosphere. After 96 h, the cells were re-stimulated
for 5 h with 1X T cell stimulation cocktail (plus protein transport
inhibitors) and then were harvested and stained with fluorescent
antibodies for flow cytometric analysis.

\textbf{Flow cytometry}

Bronchoalveolar lavage fluid (BALF) was performed via delivery of 0.8 mL
of PBS intratracheally twice through a canula and centrifuged. Cells
were immediately stained by antibodies. To obtain single-cell
suspensions from lung tissues, lungs were intubated and digested for 1
hour at 37\selectlanguage{english} with intermittent pipetting every 15 min in Hank's Balanced
Salt Solution\selectlanguage{english}(HBSS, Invitrogen) media containing 1mg/mL type 1A
collagenase (Sigma) and 30\selectlanguage{greek}μ\selectlanguage{english}g/mL DNase I (Sigma) . Homogenates were
passed through a 70 mm filter, treated with RBC lysis buffer, washed,
and resuspended in PBS with 1\% FBS. For Th cell differentiation
analysis, cells were restimulated with 1X cell stimulation cocktail
(plus protein transport inhibitors) (eBioscience) for 5 hours at 37
before surface and intracellular staining with appropriate cytokine
antibodies. For surface staining, cells were incubated with antibodies
cocktails for 30 min at 4. For intracellular staining, cells were fixed
and permeabilized using Fixation/Permeabilization Buffer Set
(eBioscience) according to manufacturer's protocol.
Fluorochrome-conjugated anti-mouse mAbs used in these experiments
included the following: Fixable Viability Dye eF780, CD45-BV510,
Siglec-F-AF647, CD11c-FITC, CD11b-PECY7, CD4-PECY7, IL-4-BV421,
IL-17A-AF647, IFN-\selectlanguage{greek}γ-\selectlanguage{english}FITC, IL-9-PE, CD44-FITC (Miltenyi), CD62L-APC,
CD3-PERCPCY5.5. All antibodies were purchased from BD Biosciences unless
otherwise specified. Cells were analyzed using Fortessa (BD) and FlowJo
X software.

\textbf{Data analysis}

Flow cytometry data were analyzed by CytExpert (Beckman, USA) or FlowJo
software. Statistical significance was performed by analysis of one-way
ANOVA with post hoc Holm-Sidak's test or a two-sided unpaired Student's
t-test. Statistical analysis was performed using Graphpad Prism 8.0
(Graphpad software, USA). All data were presented as mean\selectlanguage{ngerman}±SD. P~values
less than~0.05 were considered statistically significant.

\textbf{Results}

\textbf{IL-25 and HDM co-operated to upregulate HLA-DR, PD-L1 and OX-40L
expression on eosinophils from allergic asthma patients}

Effective antigen presentation requires expression of both MHC-II and
co-stimulatory molecules on APCs, so we firstly examined whether IL-25
had an impact on the expression of those co-stimulatory molecules of
eosinophils from allergic asthmatics in vitro. When stimulated with both
HDM and IL-25 for 18 h, the expression of HLA-DR, PD-L1 and OX-40L of
cultured eosinophils was found to be significantly increased compared
with control group. HLA-DR expression was also elevated in HDM+IL-25
group compared to HDM or IL-25 alone group. The expression of PD-L1 was
significantly higher when cultured with HDM+IL-25 than HDM alone (Figure
1).

\textbf{IL-25 and HDM co-sensitized eosinophils promoted Th2
differentiation of autologous Naïve CD4+ T cells from allergic asthma
patients}

Eosinophils were pulsed with HDM and/or IL-25 for 18 h, and then went
through a coculture procedure with autologous Naïve CD4+ T cells for up
to 48 h. Intracellular cytokines expression was tested by flow
cytometry. Signature effector cytokines IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-4, IL-9 and IL-17A
were selected to stand for Th1, Th2, Th9 and Th17 cells, respectively.
When treated with both IL-25 and HDM, eosinophils further promoted
differentiation of autologous Na\selectlanguage{ngerman}ïve CD4+ T cells towards Th2 cells,
which was unseen in IL-25 or HDM mono-stimulation group. Comparatively,
in groups where eosinophils were absent, IL-25 and HDM separately were
unable to promote Th cell differentiation effectively (Figure 2).

\textbf{IL-25 exposure induced eosinophil recruitment in vivo}

IL-25 was shown in previous research to directly activate conventional
DCs in vivo\textsuperscript{23}. Thus, we sought to determine the effect
of IL-25 in vivo on eosinophils. We administered recombinant murine
IL-25 (1\selectlanguage{greek}μ\selectlanguage{english}g/mouse) or PBS to WT mice. After 24 h, lungs were digested and
analyzed for infiltrated lung cell differentiation (Figure 3A). There
were significantly more eosinophils in lungs of IL-25-treated mice
compared with PBS-control mice (Figure 3B,3C). Thus, IL-25 promotes
eosinophils to recruit to lungs in vivo.

\textbf{IL-25 was required for induction of allergic airway
inflammation}

To determine whether IL-25 is essential for induction of allergic airway
inflammation, we subjected wild-type and IL-25-deficient mice
(Il25\textsuperscript{-/-)} to allergic airway inflammation induced by
house-dust mite (HDM), the most common allergen of allergic asthma in
Chinese patients\textsuperscript{24} (Figure 4A). After a sensitization
and challenge phase with HDM, wild-type mice developed a severe lung
pathology, increased serum IgE, and high Th2-cell-mediated inflammatory
responses. We found that Il25\textsuperscript{-/-} mice, compared with
WT mice, had an alleviated airway pathology via H\&E staining and
periodic acid-Schiff (PAS) assay (Figure 4C), decreased IgE production
(Figure 4B) and cellular infiltration in the airways (Figure 4D), less
eosinophils (Figure 4E) and reduced Th2-cytokines level, including IL-4,
IL-5, IL-13 (Figure 4F\textasciitilde{}H). These data suggest that IL-25
is required to maintain disease severity during allergen-mediated immune
responses.

\textbf{IL-25 deficiency reduced eosinophil chemotaxis after HDM
sensitization in lung and BALF}

Previous studies have identified eosinophils process antigen to promote
Th2 cells expansion\textsuperscript{25-27}. Since the antigen-presenting
role occurs during the sensitization phase, we wondered whether IL-25
affects eosinophils during the sensitization phase. Sensitized dose of
HDM was administrated intranasally into WT mice and we explored lung
eosinophils chemotaxis from day 0 to day 5 (Figure 5A) and found that on
the third day eosinophils were significantly increased after HDM
sensitization, suggesting it may exert an antigen-presenting role at
this time (Figure 5B,5C). To determine whether IL-25 contributes to the
role of eosinophils in sensitization response, we analyzed eosinophils
chemotaxis in IL-25\textsuperscript{-/-} mice 3 days after HDM
sensitization (Figure 5D). IL-25\textsuperscript{-/-} mice exhibited
reduced eosinophils compared with WT mice, both in lung and BALF (Figure
5E\textasciitilde{}H), which demonstrated the effect of IL-25 in
promoting eosinophils chemotaxis to airway to exert its role in the
sensitization phase, before the onset of effector stage.

\textbf{IL-25 promoted antigen uptake by bone marrow-derived eosinophils
in vitro}

Considering the predominant role for antigen uptake in APC, we looked at
the capacity of BmEOS to take up HDM on exposure to IL-25. We generated
eosinophils from bone marrow of mice (Figure 6A) and identified that
BmEOS become mature and reached at high purity after day 10 (Figure
6B\textasciitilde{}D). To assess whether IL-25 modulated the ability of
eosinophils to uptake antigen directly, we treated BmEOS with DQ-OVA
when pulsed with or without HDM. When pulsed with HDM, the uptake
ability of BmEOS slightly raised but without significant statistical
difference compared with control group. Pulsed with HDM and IL-25
increased significantly uptake of DQ-OVA by BmEOS, as compared to pulsed
with HDM alone (Figure 6E). These data suggested that IL-25 promoted the
ability of eosinophils to take up the antigen.

\textbf{IL-25 facilitated Th2 cell differentiation induced by
eosinophils}

To further investigate the effect of IL-25 on eosinophils in initiating
Th cell responses, BmEOS from WT mice and na\selectlanguage{ngerman}ïve CD4+ T cells from
spleens were co-cultured in the absence or presence of HDM and IL-25
(Figure 7A\textasciitilde{}B). The results showed that the percentage of
CD4+ IL-4+ Th2 cells was significantly increased in BmEOS-T co-culture
system with HDM plus IL-25 treatment, as compared with HDM treatment
alone. When culturing naïve CD4+T cells alone in the presence of HDM and
IL-25, there were no detectable differences in the percentage of CD4+
IL-4+ Th2 cells among vehicle-treated, HDM-treated and HDM plus
IL-25-treated T cells, suggesting that HDM plus IL-25 treatment did not
lead to CD4+ Th2 cells-intrinsic differentiation (Figure 7C). In
addition, HDM-treated co-cultures developed an increase in the
percentages of CD4+IFN-\selectlanguage{greek}γ\selectlanguage{english}+Th1 cells and CD4+IL-9+Th9 cells than
vehicle-treatment group. However, there were no significantly
differences in the proportions of Th1, Th9 and Th17 cells between HDM
treatment and HDM plus IL-25 treatment group, no matter in BmEOS-T
co-culture or cultures of T cells alone (Figure 7D). These led to the
conclusion that IL-25 could promote eosinophil-induced Th2 cell
polarization.

\textbf{Discussion}

Here we show that IL-25 has an important role in type 2 allergic
immunity through boosting the ability of eosinophils to initiate Th2
cells. Our data demonstrated that IL-25 promotes not only eosinophils
migration to lung, but antigen uptake by eosinophils, along with
eliciting the expression of surface costimulatory on eosinophils, which
together activate na\selectlanguage{ngerman}ïve CD4+ T cells polarization to Th2 cells,
resulting in allergic airway inflammation ultimately.

Previous studies showed that IL-25 is critical in type 2 immune response
by stimulating Th2 and ILC2 cells\textsuperscript{28, 29}. Using an in
vivo mouse model of airway inflammation, we showed that
Il25\textsuperscript{-/-}mice displayed mitigated inflammatory responses
and Th2 responses in HDM-induced asthma model, which was consistent with
the previous OVA-induced studies or in anti-IL-25 administrated mice
model\textsuperscript{28, 30}. Our results stressed the importance of
IL-25 in allergic airway inflammation again. The contribution of IL-25
on asthma symptoms by its direct action on~lung endothelial cells, DCs
and Th2 cells has been well documented\textsuperscript{28, 31-33}.
During allergic airway inflammation, the receptor for IL-25 was shown to
be expressed by eosinophils, suggesting a direct function of IL-25 on
it\textsuperscript{34}. Diverse functions of IL-25 on eosinophils have
been elucidated, including prompting cell activation, cytokine release
and survival maintenance\textsuperscript{12}, enhancing its role as
effector cells in immunity responses.

It is now clear that eosinophils are more than just terminally
differentiated effector cells\textsuperscript{35} and have
antigen-presenting cells-like functions, such as expression of
co-stimulatory molecules\textsuperscript{36}, migration toward regional
lymph nodes\textsuperscript{37}and assistant in the priming of naïve T
cells to initiate Th2 responses\textsuperscript{7, 38, 39}. The evolving
recognition of the capacity of eosinophils to engage in interaction with
CD4 lymphocytes and other immunologic cells suggests that eosinophils
could function as antigen-presenting cells to elicit antigen-specific
responses, which places eosinophils upstream of Th2 cell-mediated
effector responses\textsuperscript{40, 41}.

Antigen present cells must be able to internalize foreign antigen
efficiently and coordinately regulate antigen uptake, migration and T
cell stimulatory capacity\textsuperscript{42}. Antigen uptake and
process is an important step in the initiation of antigen-specific
responses. Enhanced allergen capturing and processing by airway APC
populations increases T cell activation and contributes to allergic
airways disease\textsuperscript{43}. In our study, IL-25 significantly
promoted antigen uptake by eosinophils, supporting the idea that IL-25
can directly modulate antigen present capability of eosinophils. We here
meanwhile investigated that IL-25 is implicated in the priming and
activation of eosinophils on their accumulation to lung. We also found
that IL-25 treatment upregulated cell surface expression of HLA-DR,
PD-L1 and OX40L on eosinophils in peripheral blood from allergic asthma
patients. PD-L1, a co-stimulatory molecule of the CD28/B7 family, is
highly expressed on dendritic cells and upregulates upon antigen uptake.
PD-L1 plays a crucial role in migration and activation of antigen
present cells\textsuperscript{44}. OX40L is the tumor necrosis factor
receptor (TNFR) superfamily, expressed on professional and
non-professional APC. It has been shown that interaction between
membrane OX40L on APCs and OX40 on naive T cells contributes to the
induction T cells polarization into the Th2 subset\textsuperscript{45,
46}. It was reported in several studies that eosinophils could express
CD40, CD80 and CD86\textsuperscript{47, 48}, but we did not observe any
significant increase in the expression of these surface molecules upon
IL-25 stimulation, which indicates probably the role of IL-25 in
promoting the antigen presentation of eosinophils is independent of
these molecules.

In co-culture studies aimed at understanding the role of IL-25 on the
antigen present capacity for eosinophils toward T cells, we determined
that IL-25 activated-eosinophils promote T cells differentiation only to
Th2 cell polarization but not Th1, Th17 and Th9 cells from naive CD4+ T
cells. When T cells culture alone, it hasn't seen IL-25 activated
directly to T cells, which is consistent with that reported by Yui-Hsi
et al, in which they observed that IL-25 alone could induce neither Th2
polarization nor phenotypic changes of Th2 memory cells, but could
induced the expanded Th2 cells driven by DCs\textsuperscript{49}.
Besides, we have observed that when HDM was added alone in EOS-T from
asthmatics co-cultures system, eosinophils were not capable to induce T
cells polarization. This result is consistent with the previous study
that human peripheral blood eosinophils pulsed with HDM drove Th cell
proliferation but the cytokine responses were not biased towards any
specific Th subset\textsuperscript{50}. However, these results differ
slightly in co-culture of mouse derived EOS and spleen T cells, in which
HDM pulsed eosinophils promote Th1 and Th9 polarization. This
discrepancy may be explained by species difference and technical
differences in the manner of culturing such as mouse bone-marrow derived
eosinophils culture with SCF, FLT and rmIL-5 in the process prior to
co-culture with T cells.

Our study suggested that IL-25 can facilitate naïve CD4+T cells
differentiation to Th2 cells as we demonstrated employing mouse-derived
cells and human peripheral blood eosinophils both. However, we were
unable to obtain eosinophils from human airway or local lung tissues to
prove it directly. Although we have uncovered multiple evidences for
IL-25 enhancing eosinophils capacity in antigen presentation, the
powerful role of DC as the classic antigen-presenting cell during airway
inflammation is still the main pathway in initiating Th2 response.
Further studies are required to define the relationship in antigen
presenting function of eosinophils with or without the condition of
excluding DCs in vivo.

In summary, we demonstrated a novel role of IL-25 in enhancing
eosinophils antigen presenting function in allergic airway inflammation.
Identifying diverse roles of eosinophils playing in the immune responses
and its related triggers provides important insights and precise targets
to eosinophil-associated diseases.

\textbf{Abbreviations}

APC: Antigen present cell; BALF: Bronchoalveolar lavage fluid; BM: Bone
marrow; BmEOS: Bone marrow derived eosinophils; Der p: Dermatophagoides
pteronyssinus; DC: Dendritic cell; EOS: Eosinophil; HDM: House dust
mite; IL: Interleukin; Il25\textsuperscript{-/-}: IL-25-deficient;
MHCII: Class II major histocompatibility complex; MLN: Mediastinal lymph
node; Th2: T helper Type 2; WT: Wild type

\textbf{Funding}

Supported by the National Natural Science Foundation of China
(81870021); Shanghai Municipal Key Clinical Specialty (shslczdzk02202);
National Key R\&D Program of China (2018YFC1311900); Shanghai Municipal
Commission of Health and Family Planning project (201740032)

\textbf{Conflict of interest}

The authors declare that they have no relevant conflicts of interest.

\textbf{AUTHOR CONTRIBUTIONS}

BP and LS conducted most of the experiments, completed the data and
contributed equally to this work. Meng Zhang offered assistance in
animal model preparation and experimental operation details; Huacheng
Yan was involved in patient related experiments including specimen
acquisition, handling and analysis; Guochao Shi made substantial
contributions to the design and conduct of the experiment. Zhenwei Xia
provided the indispensable experimental platform and constructive
guidance. WT and Ranran Dai conceived the project and led the study
design, supervised the study, reviewed the manuscript and provided final
confirmation of the revised version to be published. All authors have
read and approved the final version of manuscript.

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\textbf{FIGURE LEGEND}

\textbf{Figure 1. IL-25 and HDM co-promote expression of surface HLA-DR,
PD-L1 and OX-40L on eosinophils.}

(A) Gated on viable eosinophils
(Zombie\textsuperscript{-}CD16\textsuperscript{-}Siglec-8\textsuperscript{+}cells),
expression rate of CD40, HLA-DR, CD80, CD86, PD-L1 and OX-40L in the
presence or absence of HDM and/or IL-25 was tested by flow cytometry.
(B) Graphs showed expression rate of CD40, HLA-DR, CD80, CD86, PD-L1 and
OX-40L on eosinophils from allergic asthma patients.
n=14,\textsuperscript{**}p\textless{}0.01,\textsuperscript{*}p\textless{}0.05.

\textbf{Figure 2. IL-25 plus HDM-sensitized eosinophils promote Th2
differentiation of autologous Naïve CD4\textsuperscript{+} T cells from
allergic asthma patients.}

(A) Gated on viable CD4\textsuperscript{+} T cells
(Zombie\textsuperscript{-}CD3\textsuperscript{+}CD4\textsuperscript{+}cells),
expression of intracellular IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-4, IL-9, IL-17A were tested by
flow cytometry. (B) Graphs show percentages of intracellular IFN-\selectlanguage{greek}γ,
\selectlanguage{english}IL-4, IL-9, IL-17A expression on CD4\textsuperscript{+} T cells in
coculture system or cultured alone. n=5,
\textsuperscript{*}p\textless{}0.05.

\textbf{Figure 3. IL-25 induces eosinophils recruitment in vivo.}

Mice were instilled intranasally with 100ug/40\selectlanguage{greek}μ\selectlanguage{english}L murine recombinant
IL-25 or 40\selectlanguage{greek}μ\selectlanguage{english}L PBS. After 1 day, mice were sacrificed for analyzing lung
eosinophils. Schematic representation as in (A). (B) Flow plots showing
the extent of eosinophils (viable CD45+SiglecF+CD11c-) infiltration into
the lungs of mice after IL-25 exposure. (C) Quantification of
eosinophils infiltration after IL-25 exposure. Each dot represents a
mouse. n=5, **p<0.01, Unpaired Student's t test.

\textbf{Figure 4. IL-25 deficiency ameliorates HDM-Induced airway
inflammation.}

(A) Schematic representation of house dust mite (HDM)-induced allergic
airway inflammation of wild-type and global
IL-25\textsuperscript{-/-}mice.

(B) Serum IgE level after HDM or PBS challenge.

(C) HE\&PAS lung histology images of wild-type and global
IL-25\textsuperscript{-/-} mice during PBS or HDM challenge. Scale bars:
50 \selectlanguage{greek}μ\selectlanguage{english}m

(D) Absolute cellularity of total cells in BALF of mice in (A).

(E) Flow plots showing the percentage (left) and qualification (right)
of BALF eosinophils (CD45+SiglecF+CD11c-) infiltration into airways
after HDM or PBS challenge.

(F-H) BALF IL-4(F), IL-5(G), IL-13(H) cytokine concentration,
respectively, in wild-type and global IL-25\textsuperscript{-/-} of HDM
challenged mice.

n= 4\textasciitilde{}5, ANOVA (C, D, E, F, G and H) , *p<0.05 **p<0.01
****p<0.0001

\textbf{Figure 5. IL-25 deficiency reduces eosinophils chemotaxis after
HDM sensitization.}

(A) Schematic representation of HDM-primed eosinophils chemotaxis for
focusing on the early sensitization period.

(B-C) Flow plots showing the percentage (B) and qualification (C) of
eosinophils infiltration into lungs after HDM or PBS sensitization of WT
mice at indicated times.

(D) Schematic representation of mice 3 days after HDM-priming.

(E-H) Flow plots showing the percentage and qualification of eosinophils
infiltration into lungs (E, F) and airways (G, H) of WT and
Il25\textsuperscript{-/-} mice 3 days after HDM or PBS sensitization.
n=4 for F; n=6 for H

Unpaired Student's t test (C, D, E, F, G and H) , ns, no significant
difference, *p<0.05 **p<0.01 ***p<0.01

\textbf{Figure 6. IL-25 promotes antigen uptake by BmEOS.}

(A) Flowchart of isolation and culture of BmEOS.

(B-D) Identification of BmEOS. Histogram of Siglec-F expression at
indicated times of mouse BmEOS culture(B). Flow cytometry quantification
of Siglec-F positive BmEOS using isotype control and anti-Siglec-F
AF647-conjugated antibody(C). Light microscopic image of BmEOS at
culture day 11, Diff-Quick stain(D).

(E) At day 10\textasciitilde{}13, BmEOS were treated with or without HDM
in the absence or presence of IL-25 for 48 h. During the last 12 hours,
DQ-OVA was added. The percentage of DQ-OVA+ BmEOS and MFI of DQ-OVA were
analyzed by flow cytometry. Representative graphs, statistical data and
MFI values are shown. Cells with DQ-OVA incubated at 4 were set as
isotype.

n=4, Unpaired Student's t test (E) , *p<0.05, **p<0.01

\textbf{Figure 7. IL-25-activated Eosinophils promote Th2 polarization
in vitro.}

(A) Schematic diagram of isolated na\selectlanguage{ngerman}ïve CD4+ T cells from mice and
co-culture of T-BmEOS system.

(B) Flow cytometry identification of isolated naïve CD4+T cells (viable
CD45+CD3+CD4+ CD62L+CD44-).

(C-D) Purified naïve CD4+ T cells and BmEOS were co-cultured for 96 h
with HDM in the presence or absence of IL-25. Naïve CD4+ T cells alone
were also cultured. The differentiation of T subsets was detected by
flow cytometry.

n=3, Unpaired Student's t test (C,D) , *p\selectlanguage{english}<0.05.\selectlanguage{english}
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