Interleukin-25 and eosinophils progenitor cell mobilization in allergic asthma

Background Eosinophil-lineage committed progenitor cells (EoP) migrate from the bone marrow and differentiate locally to provide an ongoing source of mature eosinophils in asthmatic inflammatory responses in the airways. Sputum levels of EoP are increased in asthmatics compared to normal controls suggesting an exaggerated eosinophilopoietic environment in the airways. Understanding what factors promote EoP traffic to the airways is important to understand the diathesis of asthma pathology. Interleukin (IL)-25, is an epithelial-derived cytokine that promotes type 2 inflammatory responses. We have previously shown that levels of IL-25 and expression of the IL-25 receptor (IL-17RA and IL-17RB) on mature eosinophils are greater in allergic asthmatics compared to atopic non-asthmatics and non-atopic normal controls. In addition, these levels were increased significantly increased following allergen inhalation challenge and physiologically relevant levels of IL-25 stimulated eosinophil degranulation, intracellular IL-5 and IL-13 expression and primed migration to eotaxin. The current study, examined the role of IL-25 on allergen-induced trafficking of EoP in atopic asthmatics. Methods Asthmatics (n = 14) who developed allergen-induced early and late responses were enrolled in the study. Blood was collected at pre- and 24 h post-challenge. At each time point, surface expression of IL-17RA and IL-17RB on EoP was evaluated by flow cytometry. Migration assays examined the effect of IL-25 on EoP chemotactic responses, in vitro. In addition, IL-25 knockout ovalbumin (OVA) sensitized and challenged mice were studied to evaluate in vivo mobilization effects of IL-25 on newly formed EoP and mature eosinophils. Results There was a significant increase in numbers of blood EoP expressing IL-17RB, 24 h post-allergen inhalation challenge in allergic asthmatics. Pre-exposure to IL-25 primed the migrational responsiveness of EoP to stromal cell-derived factor 1α. In OVA-sensitized mice, knocking out IL-25 significantly alleviated OVA-induced eosinophil infiltration in the airway and newly formed eosinophils were reduced in the lung. Conclusions The findings of this study indicate a potential role for IL-25 in allergen-induced trafficking of EoP to the airways and local differentiation promoting tissue eosiniophilia in asthmatic responses. Electronic supplementary material The online version of this article (10.1186/s13601-018-0190-2) contains supplementary material, which is available to authorized users.


Background
Asthma is a chronic disease of the airways characterized by reversible airflow obstruction, airway inflammation and airway hypperesponsiveness. Tissue eosinophilia and type 2 cytokine producing cells including T-helper (Th) 2 cells and group 2 innate lymphoid cells, are the predominant components of the airway inflammatory cell infiltrate in subjects with allergic asthma [1].
Interleukin-25 (IL-25; IL-17E) is a pro-inflammatory cytokine that belongs to the IL-17 cytokine family and, unlike other members of the IL-17 family, plays a pivotal role in the maintenance of type 2 immune responses [2]. IL-25 has been shown to directly activate eosinophils, by up-regulation of the adhesion molecule ICAM-1, stimulate the release of pro-inflammatory chemokines such as monocyte chemoattractant protein-1, IL-8, macrophage

Open Access
Clinical and Translational Allergy *Correspondence: sehmir@mcmaster.ca inflammatory protein-1 and IL-6, as well as delay apoptosis [3,4]. The IL-25 receptor consists of two subunits, IL-17RA (the signaling sub-unit) and IL-17RB (the specific cytokine binding subunit) that form the functional heterodimeric receptor, IL-17RA/RB [5]. In a previous baseline cross-sectional study, we have shown significantly increased expression of IL-17RA and IL-17RB on mature eosinophils and plasma levels of IL-25 in asymptomatic mild allergic asthmatics compared with atopic non-asthmatics and non-atopic normal subjects [6]. In addition, we reported significant increases in plasma levels of IL-25 and intracellular IL-25 eosinophil levels, as well as IL-17RA/RB and IL-17RB receptor expression on mature eosinophils, 24 h following allergen-inhalation challenge in allergic asthmatics [7]. Furthermore in vitro experiments showed that at physiologically relevant concentrations, IL-25 stimulated eosinophil degranulation and primed the migrational responses of mature eosinophils.
A considerable body of evidence supports the view that in allergic asthma, eosinophil-lineage committed progenitor cells (EoP) traffic from the bone marrow to the lungs via the peripheral circulation and that the local tissuedriven differentiation of these cells may contribute to the development and maintenance of tissue eosinophilia [8]. The effect of IL-25 on the traffic of bone marrow-derived hemopoietic progenitor cells (HPC) and more specifically eosinophil-lineage commmited progenitor cells (EoP) to the lungs in allergic asthmatic responses has not been reported to date.
In this study, we examined IL-25 and IL-25R expression on EoP in asthmatic subjects following allergen-inhalation challenge. In addition, we employed an OVAsensitized mouse model to investigate whether traffic of mature eosinophils and newly formed eosinophils to the site of inflammation was influenced by IL-25.

Study design
Fourteen subjects with mild allergic asthma, aged between 19 and 52 years, were enrolled in the study. All volunteers were atopic with one or more positive skin prick tests; a forced expired volume in 1 s (FEV 1 ) greater or equal to 70% of predicted; and dual airway responses to inhaled allergen as determined by a fall in FEV 1 ≥ 15% within the first 2 h, followed by second fall in FEV 1 between 3 and 7 h after allergen inhalation challenge (Table 1). All subjects were steroid naïve and only intermittently used β 2 -agonists. Subjects attended the laboratory for three consecutive visits. On visit 1 (day 1), a medical history and physical examination were performed and subjects underwent a skin prick test, spirometry, methacholine inhalation challenge. On visit 2 (day 2), subjects underwent allergen inhalation challenge and spirometry was measured hourly up to 7 h post-challenge. At Visit 3 (day 3), spirometry and methacholine challenge was performed 24 h after inhalation challenge. Flow cytometric assessments were performed on blood samples collected before and 24 h post-allergen challenge. All subjects gave written informed consent, and the study was approved by the Hamilton Health Science Research Ethics Board (HIREB # 12-583).

Allergen inhalation challenge
Allergen inhalation was performed as previously described [9]. The allergen producing the largest diameter skin wheel was diluted in saline for inhalation. The concentration of allergen required to achieve a 20% decrease in FEV 1 (the allergen PC 20 ) was predicted using the methacholine PC 20 and the titration of allergen determined from the skin prick test. The early asthmatic response (EAR) was recorded as the greatest fall in FEV 1 between 0 and 2 h after allergen inhalation, whereas the greatest drop in FEV 1 between 3 and 7 h was recorded as the late asthmatic response (LAR) as previously described [9].

Bronchoalveolar lavage fluid (BALF), blood and bone marrow sampling
BALF collection was performed 24 h after the final OVA or PBS challenge. Lungs were lavaged with 1 mL PBS through the trachea and BALF was collected. Blood (0.5-0.8 mL) was drawn prior to BALF collection into heparin from each mouse. Bone marrow was harvested from the femur and tibia into heparin as previously described [12].
Smears of blood and bone marrow samples were made to perform eosinophil counts following standard staining with hematoxolin and eosin.

Measurement of eosinophil number in BALF
Cells were seeded in PBS medium (Beyotime Institute of Biotechnology, Haimen, China) at 1 × 10 5 cells/mL and stained with Fast Wright and Giemsa Stain kit (Nanjing Jiancheng Technology Co., Ltd., Nanjing, China), according to manufacturer's protocol. Eosinophils were counted with a light microscope and expressed as percent eosinophils.

Inflammatory index measurement
The lung inflammation index score after OVA or diluent challenge was measured as previously described [13]. The index is: 0 equals no inflammatory infiltration; 1 equals a minimal inflammatory cell infiltration; 2 equals a layer of inflammatory cells annular infiltration; 3 equals 2-4 layers of inflammatory cell ring infiltration; 4 equals more than 4 layers of inflammatory cells annular infiltration.

Immunohistochemistry (IHC) semi-quantitative analysis
The expression levels of BrdU in lung tissues were quantified by Image-Pro Plus 6.0, expressed as mean optical density (OD), which equals intensity of optical density divided by area of lung tissues observed under microscope (400×). Newly produced eosinophils were identified as BrdU positive and eosin positive cells.

Statistical analysis
Statistical analysis was performed using GraphPad Prism5 software (GraphPad Software Inc.). This study is powered on sputum eosinophil progenitor cells in mild asthmatics, and changes 24 h post-allergen were the primary outcome. Assuming within subject variability from previously published data in mild asthmatics [9], the sample size required to detect the "minimal important differences" between baseline and 24 h post-allergen measurements was calculated. Based on repeated measures ANOVA analyses, using the sample size module of NCSS statistical package with β = 0.20 (power = 80%) and α = 0.05 (likelihood of type 1 error = 5%) and a minimum important difference = 0.72, SD = 0.59, the sample size is calculated to be n = 14. Normally distributed data are expressed as mean ± SEM. The methacholine PC 20 is expressed as geometric mean and geometric standard error of the mean (GSEM). For HPC and EoP IL-25 receptor expression and migration experiments, a repeated measures ANOVA was used. Post-hoc comparisons were performed using the Tukey's multiple comparison tests. p < 0.05 was considered significant for all analyses.

Discussion
This study has demonstrated that following allergeninhalation challenge in allergic asthmatic subjects, IL-17RB is up-regulated on the surface of HPC, as well as EoP in the blood. In addition, IL-25 primes migrational responses of blood-derived HPC and EoP to the progenitor chemoattractant, SDF-1α. These findings suggest that in response to inhaled allergen, upregulation of IL-25 receptor binding sub-unit expression on EoP in peripheral blood may promote increased occupancy of IL-25 on its specific receptor and stimulate priming of migrational responsiveness, thereby facilitating the homing of eosinophil precursors to the airways. Furthermore, in OVA-sensitized mice, knocking out IL-25 significantly alleviated lung inflammation, airway eosinophil infiltration and lung homing of newly produced eosinophils. The biological effects of IL-25 are mediated by IL-25 receptor, which is composed of sub-units IL-17RA and IL-17RB. IL-25 is the high affinity ligand for IL-17RB, while IL-17RA shares the common ligand for IL-17A [5,14]. Polymorphisms in the IL-17RB gene in humans have been linked with asthma susceptibility [15]. In a previous study, we have shown that IL-25 receptor expression on eosinophils is markedly higher in allergic asthmatics compared with atopic non-asthmatic and normal subjects [6]. In addition, we showed that the level of plasma IL-25 significantly increases following allergen inhalation challenge in allergic asthmatics [7]. In humans, IL-25 is produced by structural cells, such as epithelial and endothelial cells, and inflammatory cells, such as eosinophils, basophils and mast cells. IL-25 has been shown to link innate and adaptive immunity by enhancing type-2 cytokine production, including IL-5 and IL-13 [2].
The current study suggests that IL-25 plays a role in the recruitment of immature eosinophils to the airways in asthma. Our previous research has shown that EoP traffic from the systemic circulation into inflamed tissue sites, the migration orchestrated by locally produced chemokines, such as SDF-1α [16]. In line with these findings, our current data demonstrate that, although IL-25 did not directly stimulate migrational responses of EoP, pre-exposure to IL-25 enhanced the subsequent migrational response to SDF-1α. As such, it can be postulated that IL-25 may contribute to eosinophilic inflammation observed in the lung following allergen exposure through the priming of migrational responses of immature and mature eosinophils.
We have previously shown a significant increase in HPC and EoP in the sputum 24 h post-allergen challenge, which was associated with a significant increase in the expression of receptors for epithelial derived cytokines including TSLP (TSLPR and CD127) and IL-33 (ST2) on HPC and EoP [10]. Furthermore, pre-exposure to TSLP and IL-33 primed the migration of progenitor HPC and this effect was inhibited by blocking antibodies to TSLPR and ST2, respectively, suggesting that lung-homing of HPC maybe orchestrated by epithelial-derived cytokines, including TSLP and IL-33 [10]. Our current data support the view that the epithelial-derived cytokine IL-25 can prime the migrational response of these cells and promote lung-homing. In contrast to findings with TSLP and IL-33, we show here for the first time that IL-25 can prime the migrational responses of HPC and EoP while the latter cytokines only had effects on HPC [10]. A limitation of the study was that we performed these receptor up-regulation analyses in the blood as opposed to sputum samples as have been described in the above mentioned study. However, our findings in blood-derived eosinophil progenitor cell populations were similar to changes in sputum. Furthermore, the priming experiments with IL-25 were in agreement with the priming experiments performed with TSLP and IL-33 in blood derived cells suggesting similarity in the underlying mechanism.
The pro-inflammatory effects of IL-25 has been well demonstrated in animal models. Exogenous administration of IL-25, or transgenic expression induces type 2 asthma-like inflammation in the airways in mice [17,18]. Conversely, anti-IL-25 antibody reduces airway inflammation in animal models of allergic asthma [19,20]. In addition, IL-25-deficient mice have significant suppression of the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids (BALF) [21]. In this current study, OVA challenge of sensitized IL-25-deficient mice not only decreased mobilization of mature eosinophils, but also newly formed eosinophils. In IL-25 KO mice, the attenuation of newly produced eosinophils (BrdU + eosinophils) was observed the airways (BALF) and bone marrow samples suggesting that IL-25 may be involved in the formation of newly produced eosinophils in the bone marrow, as well as in the airways. We acknowledge that by labeling with BrdU, this study only enumerated newly formed eosinophils and not EoP per se. As such, it is unclear as to whether these newly formed eosinophils matured within the bone marrow and migrated to the airways, or if EoP migrated to the airways and differentiated locally within the tissue to mature eosinophils. However, we have previously shown that EoP traffic to the site of inflammation and have the potential of forming eosinophils in situ [22,23] thus supporting the view that the BrDU + eosinophils arose as a result of local differentiative processes.

Conclusions
In summary, IL-25 high affinity receptor part (IL-17RB) expression on EoP is increased in the peripheral blood of subjects with asthma after allergen challenge. IL-25 also enhanced the migrational response of eosinophil progenitors. IL-25 knockout mice showed decreased eosinophilic inflammation in the bone marrow and airways. Percentage of BrdU positive eosinophils of BALF, blood and bone marrow from wild type and IL-25 KO mouse models that were sensitized and challenged with OVA or PBS (control) (*p < 0.05, **p < 0.01 and ***p < 0.001, n = 5) Fig. 6 Expression of BrdU measured by immunohistochemistry wild type and IL-25 KO mouse models that were sensitized and challenged with OVA or PBS (control) (*p < 0.05, **p < 0.01 and ***p < 0.001, n = 5)