IL-25 Promotes Eosinophils Antigen Presenting Function in Allergic Asthma

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. Objective: To elucidate IL-25 on eosinophils antigen presenting function during allergic asthma and its related mechanism. 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 naïve 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 -/- 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. 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 -/- 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. 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. IL-25 Promotes Eosinophils Antigen Presenting Function in Allergic Asthma Running Title：IL-25 facilitates eosinophils APC capacity Bo Peng 1,2 *, Lin Sun 1,2 *, Meng Zhang 3, Huacheng Yan 1,2, Guochao Shi 1,2, Zhenwei Xia 3 †, Ranran Dai 1,2 †, Wei Tang 1,2 † 1. Department of Respiratory and Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,197, Rui Jin Er Road, Shanghai, China. 2. Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases, Shanghai, China. 3. Department of Pediatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197, Rui Jin Er Road, Shanghai, China. *, † These authors have contributed equally 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: [email protected]. 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: [email protected]. 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: [email protected]. Phone number: +86-21-6437004

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

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.

To elucidate IL-25 on eosinophils antigen presenting function during allergic asthma and its related mechanism.

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 naïve 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 -/- 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.

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 -/- 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.

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.

IL-25, eosinophil, APC, allergic asthma

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 1, 2 . The most predominant and clearly described endotype of asthma is eosinophilic asthma, which accounts for over 50% of adults with asthma 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) 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 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 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 10, 11 . Although the diverse roles of IL-25 on eosinophils including enhancing survival or reducing apoptosis have been described 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 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 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. 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>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. In vitro eosinophils stimulation assays and co-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 17 . Purity of isolated cells was checked by flowcytometry and was always >95%. Purified eosinophils were resuspended at 1.0x10 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 (100μg/ml, Greer Lab, low endotoxin) and/or IL-25(1ng/ml, Peprotech) at 37°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 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 25μl/ml ImmunoCult Human CD3/CD28 T Cell Activator (Stem Cell). Cells were cultured at 37°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-γ 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°C in the dark, and then washed and resuspended in FACS buffer for analysis. All antibodies were purchased from Biolegend unless otherwise specified. Mice IL-25-deficient mice（Il25 -/- ）were obtained from Tsinghua university, which have been described in the previous studies 18, 19 . The genotyping primers were as follows: forward, 5′-CTGCTCCAGTCAGCCTCTCT-3′; reverse1, 5′AGCAGCTGGGCAAGTGAC-3′; reverse2,5′ AGGTGGAGAAAGTGCCTGT-3′. 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 -/- mice 6 to 8 weeks of age were mainly used for experiments. The HDM-induced mouse asthma model was established as described previously 20 . Briefly, mice were sensitized intranasally with 100 μg house dust mite extracts (HDM) (Greer Labs, low endotoxin) in 40 μL PBS on day 0 and challenged intranasally with 10 μg 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 μg 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 1μg IL-25(R&D) in 40 μL 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-γ 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. 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 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 1×10^6/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. In vitro Polarization of CD4+ T Cells with BmEOS Spleens were removed from C57bl/6J 6~8-week-old mice, mechanically disrupted, treated with RBC lysis buffer and run through 70 μm cell strainer. Suspensions were resuspended in MACS buffer, labelled with an antibody cocktail and naïve 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. 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℃ with intermittent pipetting every 15 min in Hank’s Balanced Salt Solution（HBSS, Invitrogen) media containing 1mg/mL type 1A collagenase (Sigma) and 30μg/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-γ-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. 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±SD. P values less than 0.05 were considered statistically significant.

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). 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-γ, 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 Naïve 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). IL-25 exposure induced eosinophil recruitment in vivo IL-25 was shown in previous research to directly activate conventional DCs in vivo 23 . Thus, we sought to determine the effect of IL-25 in vivo on eosinophils. We administered recombinant murine IL-25 (1μg/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. 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 -/-) to allergic airway inflammation induced by house-dust mite (HDM), the most common allergen of allergic asthma in Chinese patients 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 -/- 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~H). These data suggest that IL-25 is required to maintain disease severity during allergen-mediated immune responses. 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 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 -/- mice 3 days after HDM sensitization (Figure 5D). IL-25 -/- mice exhibited reduced eosinophils compared with WT mice, both in lung and BALF (Figure 5E~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. 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~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. 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 naïve CD4+ T cells from spleens were co-cultured in the absence or presence of HDM and IL-25 (Figure 7A~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-γ+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.

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 naïve 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 28, 29 . Using an in vivo mouse model of airway inflammation, we showed that Il25 -/- 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 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 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 34 . Diverse functions of IL-25 on eosinophils have been elucidated, including prompting cell activation, cytokine release and survival maintenance 12, enhancing its role as effector cells in immunity responses. It is now clear that eosinophils are more than just terminally differentiated effector cells 35 and have antigen-presenting cells-like functions, such as expression of co-stimulatory molecules 36, migration toward regional lymph nodes 37 and assistant in the priming of naïve T cells to initiate Th2 responses 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 40, 41 . Antigen present cells must be able to internalize foreign antigen efficiently and coordinately regulate antigen uptake, migration and T cell stimulatory capacity 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 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 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 45, 46 . It was reported in several studies that eosinophils could express CD40, CD80 and CD86 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 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 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. 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 -/- : IL-25-deficient; MHCII: Class II major histocompatibility complex; MLN: Mediastinal lymph node; Th2: T helper Type 2; WT: Wild type 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) Conflict of interest The authors declare that they have no relevant conflicts of interest. 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. REFERNCE 1. Hammad H, Lambrecht BN. The basic immunology of asthma. Cell. 2021;184(6):1469-85. 2. Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol. 2015;16(1):45-56. 3. Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. Lancet. 2018;391(10122):783-800. 4. Schuijs MJ, Hammad H, Lambrecht BN. Professional and ’Amateur’ Antigen-Presenting Cells In Type 2 Immunity. Trends in Immunology. 2019;40(1):22-34. 5. Hogan SP, Rosenberg HF, Moqbel R, et al. Eosinophils: biological properties and role in health and disease. Clin Exp Allergy. 2008;38(5):709-50. 6. Shi HZ, Humbles A, Gerard C, Jin Z, Weller PF. Lymph node trafficking and antigen presentation by endobronchial eosinophils. J Clin Invest. 2000;105(7):945-53. 7. Shi HZ, Xiao CQ, Li CQ, et al. Endobronchial eosinophils preferentially stimulate T helper cell type 2 responses. Allergy. 2004;59(4):428-35. 8. MacKenzie JR, Mattes J, Dent LA, Foster PS. Eosinophils promote allergic disease of the lung by regulating CD4(+) Th2 lymphocyte function. Journal of Immunology. 2001;167(6):3146-55. 9. Xu M, Dong C. IL-25 in allergic inflammation. Immunol Rev. 2017;278(1):185-91. 10. Tang W, Smith SG, Salter B, et al. Allergen-Induced Increases in Interleukin-25 and Interleukin-25 Receptor Expression in Mature Eosinophils from Atopic Asthmatics. Int Arch Allergy Imm. 2016;170(4):234-42. 11. Tang W, Smith SG, Beaudin S, et al. IL-25 and IL-25 Receptor Expression on Eosinophils from Subjects with Allergic Asthma. Int Arch Allergy Imm. 2014;163(1):5-10. 12. Wong CK, Cheung PF, Ip WK, Lam CW. Interleukin-25-induced chemokines and interleukin-6 release from eosinophils is mediated by p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-kappaB. Am J Respir Cell Mol Biol. 2005;33(2):186-94. 13. Cheung PF, Wong CK, Ip WK, Lam CW. IL-25 regulates the expression of adhesion molecules on eosinophils: mechanism of eosinophilia in allergic inflammation. Allergy. 2006;61(7):878-85. 14. Tworek D, Smith SG, Salter BM, et al. IL-25 Receptor Expression on Airway Dendritic Cells after Allergen Challenge in Subjects with Asthma. Am J Respir Crit Care Med. 2016;193(9):957-64. 15. Li HJ, Zhang CQ, Lu DG, et al. IL-25 Promotes Th2 Immunity Responses in Airway Inflammation of Asthmatic Mice via Activation of Dendritic Cells. Inflammation. 2014;37(4):1070-7. 16. Claudio E, Tassi I, Wang HS, Tang WH, Ha HL, Siebenlist U. Cutting Edge: IL-25 Targets Dendritic Cells To Attract IL-9-Producing T Cells in Acute Allergic Lung Inflammation. Journal of Immunology. 2015;195(8):3525-9. 17. Farhan RK, Vickers MA, Ghaemmaghami AM, Hall AM, Barker RN, Walsh GM. Effective antigen presentation to helper T cells by human eosinophils. Immunology. 2016;149(4). 18. Angkasekwinai P, Chang SH, Thapa M, Watarai H, Dong C. Regulation of IL-9 expression by IL-25 signaling. Nat Immunol. 2010;11(3):250-6. 19. Xu M, Lu H, Lee YH, et al. An Interleukin-25-Mediated Autoregulatory Circuit in Keratinocytes Plays a Pivotal Role in Psoriatic Skin Inflammation. Immunity. 2018;48(4):787-98 e4. 20. Hammad H, Plantinga M, Deswarte K, et al. Inflammatory dendritic cells–not basophils–are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J Exp Med. 2010;207(10):2097-111. 21. Dyer KD, Moser JM, Czapiga M, Siegel SJ, Percopo CM, Rosenberg HF. Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J Immunol. 2008;181(6):4004-9. 22. X T. Bone Marrow Derived Eosinophil Cultures. bio-protocol. 2014. 23. Claudio E, Wang H, Kamenyeva O, Tang W, Ha HL, Siebenlist U. IL-25 Orchestrates Activation of Th Cells via Conventional Dendritic Cells in Tissue to Exacerbate Chronic House Dust Mite-Induced Asthma Pathology. J Immunol. 2019;203(8):2319-27. 24. Li J, Sun B, Huang Y, et al. A multicentre study assessing the prevalence of sensitizations in patients with asthma and/or rhinitis in China. Allergy. 2009;64(7):1083-92. 25. Shi HZ, Xiao CQ, Li CQ, et al. Endobronchial eosinophils preferentially stimulate T helper cell type 2 responses. Allergy. 2004;59(4):428-35. 26. Shi HZ, Humbles A, Gerard C, Jin Z, Weller PF. Lymph node trafficking and antigen presentation by endobronchial eosinophils. J Clin Invest. 2000;105(7):945-53. 27. MacKenzie JR, Mattes J, Dent LA, Foster PS. Eosinophils promote allergic disease of the lung by regulating CD4(+) Th2 lymphocyte function. J Immunol. 2001;167(6):3146-55. 28. Angkasekwinai P, Park H, Wang YH, et al. Interleukin 25 promotes the initiation of proallergic type 2 responses. J Exp Med. 2007;204(7):1509-17. 29. Huang Y, Guo L, Qiu J, et al. IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential ’inflammatory’ type 2 innate lymphoid cells. Nat Immunol. 2015;16(2):161-9. 30. Tang W, Smith SG, Du W, et al. Interleukin-25 and eosinophils progenitor cell mobilization in allergic asthma. Clin Transl Allergy. 2018;8:5. 31. Kaiko GE, Phipps S, Angkasekwinai P, Dong C, Foster PS. NK cell deficiency predisposes to viral-induced Th2-type allergic inflammation via epithelial-derived IL-25. J Immunol. 2010;185(8):4681-90. 32. Gregory LG, Jones CP, Walker SA, et al. IL-25 drives remodelling in allergic airways disease induced by house dust mite. Thorax. 2013;68(1):82-90. 33. Hongjia L, Caiqing Z, Degan L, et al. IL-25 promotes Th2 immunity responses in airway inflammation of asthmatic mice via activation of dendritic cells. Inflammation. 2014;37(4):1070-7. 34. Corrigan CJ, Wang W, Meng Q, et al. Allergen-induced expression of IL-25 and IL-25 receptor in atopic asthmatic airways and late-phase cutaneous responses. J Allergy Clin Immunol. 2011;128(1):116-24. 35. Weller PF, Spencer LA. Functions of tissue-resident eosinophils. Nat Rev Immunol. 2017;17(12):746-60. 36. Wang HB, Ghiran I, Matthaei K, Weller PF. Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells. J Immunol. 2007;179(11):7585-92. 37. Duez C, Dakhama A, Tomkinson A, et al. Migration and accumulation of eosinophils toward regional lymph nodes after airway allergen challenge. J Allergy Clin Immunol. 2004;114(4):820-5. 38. Padigel UM, Lee JJ, Nolan TJ, Schad GA, Abraham D. Eosinophils can function as antigen-presenting cells to induce primary and secondary immune responses to Strongyloides stercoralis. Infect Immun. 2006;74(6):3232-8. 39. Del Pozo V, De Andres B, Martin E, et al. Eosinophil as antigen-presenting cell: activation of T cell clones and T cell hybridoma by eosinophils after antigen processing. Eur J Immunol. 1992;22(7):1919-25. 40. Weller PF. The immunobiology of eosinophils. N Engl J Med. 1991;324(16):1110-8. 41. Jacobsen EA, Zellner KR, Colbert D, Lee NA, Lee JJ. Eosinophils regulate dendritic cells and Th2 pulmonary immune responses following allergen provocation. J Immunol. 2011;187(11):6059-68. 42. Lanzavecchia A. Mechanisms of antigen uptake for presentation. Curr Opin Immunol. 1996;8(3):348-54. 43. von Garnier C, Wikstrom ME, Zosky G, et al. Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa. J Immunol. 2007;179(9):5748-59. 44. Lucas ED, Schafer JB, Matsuda J, Kraus M, Burchill MA, Tamburini BAJ. PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity. Cell Rep. 2020;33(2):108258. 45. Ito T, Wang YH, Duramad O, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med. 2005;202(9):1213-23. 46. Di C, Lin X, Zhang Y, et al. Basophil-associated OX40 ligand participates in the initiation of Th2 responses during airway inflammation. J Biol Chem. 2015;290(20):12523-36. 47. Wang HB, Ghiran I, Matthaei K, Weller PF. Airway eosinophils: Allergic inflammation recruited professional antigen-presenting cells. Journal of Immunology. 2007;179(11):7585-92. 48. Duez C, Dakhama A, Tomkinson A, et al. Migration and accumulation of eosinophils toward regional lymph nodes after airway allergen challenge. J Allergy Clin Immun. 2004;114(4):820-5. 49. Wang YH, Angkasekwinai P, Lu N, et al. IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells. J Exp Med. 2007;204(8):1837-47. 50. Farhan RK, Vickers MA, Ghaemmaghami AM, Hall AM, Barker RN, Walsh GM. Effective antigen presentation to helper T cells by human eosinophils. Immunology. 2016;149(4):413-22. FIGURE LEGEND 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 - CD16 - Siglec-8 + 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, ** p<0.01, * p<0.05. Figure 2. IL-25 plus HDM-sensitized eosinophils promote Th2 differentiation of autologous Naïve CD4 + T cells from allergic asthma patients. (A) Gated on viable CD4 + T cells (Zombie - CD3 + CD4 + cells), expression of intracellular IFN-γ, IL-4, IL-9, IL-17A were tested by flow cytometry. (B) Graphs show percentages of intracellular IFN-γ, IL-4, IL-9, IL-17A expression on CD4 + T cells in coculture system or cultured alone. n=5, * p<0.05. Figure 3. IL-25 induces eosinophils recruitment in vivo. Mice were instilled intranasally with 100ug/40μL murine recombinant IL-25 or 40μL 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. 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 -/- mice. (B) Serum IgE level after HDM or PBS challenge. (C) HE&PAS lung histology images of wild-type and global IL-25 -/- mice during PBS or HDM challenge. Scale bars: 50 μm (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 -/- of HDM challenged mice. n= 4~5, ANOVA (C, D, E, F, G and H), *p＜0.05 **p＜0.01 ****p＜0.0001 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 -/- 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 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 10~13, 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 Figure 7. IL-25-activated Eosinophils promote Th2 polarization in vitro. (A) Schematic diagram of isolated naïve 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＜0.05. Supplementary Material File (table s1.xlsx) - Download - 11.76 KB Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 347views 48downloads Citations Download citation Bo Peng, Lin Sun, Meng Zhang, et al. IL-25 Promotes Eosinophils Antigen Presenting Function in Allergic Asthma. Authorea. 31 January 2024. DOI: https://doi.org/10.22541/au.170669900.09286201/v1 DOI: https://doi.org/10.22541/au.170669900.09286201/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.