Effects of Eosinophilopoietins and C-C Chemokines on Chemotaxis, Adhesion, and ROS Production of Blood Eosinophil Subtypes in Asthma Patients

Abstract

Purpose

Two distinct blood eosinophil subtypes have recently been described as inflammatory-like (iEOS-like) and resident-like (rEOS-like) eosinophils. Eosinophilopoietins - interleukin (IL)-3, IL-5, and GM-CSF, as well as C-C chemokines - CCL11, CCL24, and CCL26 (eotaxin-1, 2, 3), CCL5 (RANTES), are key regulators of eosinophil activity. However, our understanding of the effects of these cytokines on blood eosinophil subtype chemotaxis, adhesion, and reactive oxygen species (ROS) production in asthma is limited.

Patients and Methods

The study enrolled 13 patients with non-severe allergic asthma (AA), 12 patients with severe eosinophilic asthma (SEA), and 10 healthy subjects (HS). Additionally, AA patients underwent bronchial allergen challenge with D. pteronyssinus, and all scheduled tests were repeated 24 hours later. Ex vivo blood eosinophils were purified using Ficoll gradient centrifugation and magnetic separation, subtyped by CD62L-based magnetic separation, isolating iEOS-like (CD62L⁻) and rEOS-like (CD62L⁺) cells. In vitro stimulation of the isolated eosinophils was performed using recombinant human cytokines and chemokines (IL-3, IL-5, GM-CSF; CCL5, 11, 24, 26). Eosinophil chemotaxis was evaluated using Transwell migration assay, adhesion to airway smooth muscle cells (ASMC) by eosinophil peroxidase activity assay, and ROS production measured using a dihydrorhodamine-123 fluorescent probe and flow cytometer.

Results

In all groups, IL-3, IL-5, and GM-CSF enhanced iEOS-like cells’ chemotaxis, ROS production, and adhesion to ASMC, with no impact on rEOS-like cell chemotaxis, and only GM-CSF promoted rEOS-like cell adhesion. SEA patients’ rEOS-like cells’ ROS production following activation with eosinophilopoietins was higher compared to the AA group. Allergen-activated AA patients’ blood eosinophil subtypes’ ROS production was further upregulated by IL-3, IL-5, and GM-CSF, p < 0.05, but these cytokines had differential effects on chemotaxis and adhesion to ASMC. In AA and SEA, CCL5, 11, 24 and 26 promoted chemotaxis of blood eosinophil subtypes, p < 0.05. Lastly, CCL26 had a higher effect on iEOS-like cells in AA.

Conclusion

Eosinophilopoietins and C-C chemokines increase chemotaxis, adhesion, and ROS production of blood eosinophil subtypes, notably in asthma patients. GM-CSF and CCL26 are the most potent promoters of eosinophil chemotaxis and adhesion, particularly of iEOS-like cells.

Introduction

Asthma is a chronic inflammatory respiratory disease estimated to affect more than 262 million individuals worldwide.¹ It is characterized by distinct structural and functional changes in the airways, bronchial hyperresponsiveness, and fluctuating airway obstruction.² The symptoms of asthma may periodically worsen, with exacerbations occurring due to exposure to respiratory infections, allergens, environmental pollutants, occupational irritants, or medication.³ During an exacerbation, various mediators released by airway epithelium and local tissue immune cells⁴ stimulate circulating blood inflammatory cells, including eosinophils, to migrate to the respiratory tract, where they may further release various pro-inflammatory mediators—including cationic proteins, chemokines, enzymes, cytokines, growth factors, and reactive oxygen species (ROS).⁵ These mediators may contribute to airway inflammation, tissue damage, and structural changes.⁶

Asthma is a heterogeneous disease defined by underlying immune-pathological characteristics, which can be highlighted by the presence of specific lineages of effector T-cells and innate lymphoid cells (ILC).⁷ Type 2 inflammation predominates in asthma, characterized by T helper 2 (Th2) or ILC2-dependent cytokine interleukin (IL)-4, IL-5, and IL-13 overexpression,⁸ contributing to blood and sputum eosinophilia. The elucidation of type 2 inflammatory mechanisms has led to the development of targeted antibody therapies, which have gained approval for the treatment of severe asthma.⁹ Therapeutic monoclonal antibodies targeting the IL-5/IL-5 receptor pathway have shown great results in depleting blood eosinophils and reducing exacerbations in patients with severe eosinophilic asthma (SEA).^10,11 However, treated patients still display a relatively high amount of functional lung tissue eosinophils, indicating that supplemental therapies involving different treatment targets are needed.

Eosinophil maturation, differentiation, activation, and survival are largely controlled by granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3, and, particularly, IL-5.^5,12 The receptors for these cytokines have a unique ɑ-chain, but share a common β-chain (CD131) receptor subunit, which is essential for signal transduction and may explain the overlapping activities of these cytokines.¹³ Through the engagement of this βc chain with cytokine-specific ɑ-receptors, signaling cascades are initiated, leading to eosinophil priming - potentially augmenting eosinophil adhesion and chemotactic responses, and lowering the activation threshold for NADPH oxidase-dependent ROS production.¹⁴ A Dihydrorhodamine (DHR)-123 assay was used to measure intracellular ROS production, which may reflect the oxidative stress and epithelial crosstalk driven by activated eosinophils spontaneously generating ROS during inflammation.^15,16 IL-3, IL-5, and GM-CSF, collectively known as eosinophilopoietins, play a crucial role as mediators of bodily defense and innate immunity. Still, they may also participate in inflammatory processes, including the onset and progression of chronic inflammatory airway diseases.¹⁷

Blood eosinophil recruitment into the tissue is also regulated by chemoattractants, namely C-C chemokines eotaxins-1, 2, 3 (CCL11, CCL24, CCL26), and regulated on activation, normal T cell expressed and secreted (RANTES, CCL5) via the C-C chemokine receptor (CCR)3, largely expressed by eosinophils,¹⁸ and basophils.¹⁹ Eosinophil chemotaxis is triggered by activation of surface CCR3 by ligand chemokines eotaxins-1, 2, 3. The receptor-chemokine signaling leads to the activation of β₂ integrins, specifically CD11b/CD18 on eosinophils, causing the cells to adopt an extended conformation and enabling firm adhesion to structural airway cells.^20,21 Recently, immunophenotyping studies confirmed the presence of distinct eosinophil subtypes: resident eosinophils (rEOS) and inflammatory eosinophils (iEOS) in circulation and tissues.^22–25 The robust distinction was mainly focused on CD62L expression in eosinophil subtypes, identifying iEOS as CD62L-negative, whereas rEOS expressed variable levels of CD62L. L-selectin (CD62L) is a type-I transmembrane glycoprotein and cell adhesion molecule expressed by leukocytes, including eosinophils and basophils,²⁶ and has been known to cooperate with other selectins and integrins to support leukocyte, particularly lymphocyte, rolling on inflamed vascular endothelium before firm adhesion and transmigration.²⁷ A potential functional consequence of activated eosinophils shedding CD62L is to terminate endothelial rolling and enable firm adhesion and transendothelial migration into tissues.^28,29 In addition, our recent study has found that iEOS possess elevated cytotoxic granule protein gene expression and dominate the circulating blood eosinophil population in severe asthma patients,³⁰ while subsequently increased iEOS abundance and eosinophil viability were associated with worse clinical outcomes following anti-IL-5 biologic treatment.^31,32 Therefore, identification of the major chemokines and cytokines that regulate the inflammatory properties of distinct eosinophil subtypes may provide opportunities for the development of putative asthma therapies.

This study aimed to investigate the effects of IL-3, IL-5, and GM-CSF on the adhesion of blood eosinophil subtypes to airway smooth muscle cells (ASMC), chemotaxis, and spontaneous ROS production, in addition to assessing the influence of C-C chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, and RANTES/CCL5) on eosinophil subtype chemotaxis. Moreover, we used a bronchial allergen challenge (BAC) model with house dust mite Dermatophagoides pteronyssinus (D. pteronyssinus) allergen to evaluate the activity of blood eosinophil subtypes in late-phase allergen-induced inflammatory responses. An allergen inhalation challenge is used to examine the mechanisms of allergen-induced airway response. Following an allergen attack, the early phase reaction, which reaches a maximum within 30 min and is resolved by 1–3 h, reflects immediate mast cell and mediator-driven bronchoconstriction.³³ Meanwhile, the late phase reaction (approximately 12–24 hours post-exposure) is characterized by the increase, activation, and tissue retention of inflammatory cells - particularly eosinophils - in the airways.^34,35 Investigating the biological activity and role of distinct blood eosinophil subtypes in asthma pathogenesis could offer insights into the clinical implications of eosinophil significance, treatment targets, and enhanced disease control.

Materials and Methods

Ethics

The study complies with the Declaration of Helsinki. All subjects involved were presented with the research protocol approved by the Lithuanian University of Health Sciences (LUHS) Committee of Regional Biomedical Research Ethics (BE-2-58) and signed a written consent form before participation. The study is registered in the US National Institutes of Health ClinicalTrials.gov trial registry with the unique identifier NCT04542902.

Study Design and Experimental Plan

The individuals involved in the study were all patients from the Department of Pulmonology at the LUHS Hospital Kaunas Clinics. Each subject visited the LUHS Hospital Kaunas Clinics multiple times. During their initial screening visit, all subjects underwent a health history assessment, a physical examination, an allergy skin prick test, and spirometry to confirm their eligibility for inclusion or exclusion, as shown in Table 1. Additionally, a methacholine challenge test was conducted on the AA group. If participants satisfied the criteria, they were informed about the requirements for participation in the study, and a written informed consent was signed. In total, 35 adult subjects participated in this study. The participants consisted of adult men and women, aged 18–69. Each study participant was examined according to the study design and experimental plan, depicted in Figure 1.

Table 1.

Inclusion and Exclusion Criteria of the Study

	Inclusion Criteria
Allergic asthma (AA) group:	-Diagnosed with AA -Free of inhaled steroids for ≥ 1 month before study -Positive skin prick test for at least Dermatophagoides pteronyssinus (house dust mite) allergen -Positive methacholine challenge or bronchial reversibility test
Severe eosinophilic asthma (SEA) group:	-Severe asthma history ≥ 12 months -High doses of inhaled steroids + long-acting beta agonist + episodic use of oral steroids ≥ 12 months -Uncontrolled asthma ≥ 12 months -Two or more asthma exacerbations per year, which require short-term systemic corticosteroids (≤14 days) -Peripheral blood eosinophil ≥ 0.15 × 109/L
Healthy subject (HS) group:	-No chronic respiratory or other diseases -Negative skin prick test
	Exclusion criteria
All groups:	-Age < 18 years -Asthma exacerbation ≤ 1 month before study -Supported asthma therapy of oral corticosteroid (>30 days) -Active airway infection ≤ 1 month before study -Coronavirus infectious disease (COVID-19) ≤ 1 month prior to study -Active smoking (at least one cigarette a day), former smoker (at least 100 cigarettes in lifetime) -Clinically significant non-controlled other organ disease

Abbreviations: AA, allergic asthma; HS, healthy subject; SEA, severe eosinophilic asthma.

Figure 1.

Study design and experimental plan.

Abbreviations: AA, allergic asthma; ASMC, airway smooth muscle cells; SEA, severe eosinophilic asthma; FeNO, fractional exhaled nitric oxide; Ig, immunoglobulin; iEOS, inflammatory eosinophil; IL, interleukin; rEOS, resident eosinophil; ROS, reactive oxygen species; GM-CSF, granulocyte macrophage-colony stimulating factor; DHR, dihydrorhodamine.

After inclusion in the study, the experimental visit was conducted within 0 to 4 weeks. During this visit, subjects underwent lung function testing (spirometry), fractional exhaled nitric oxide (FeNO) evaluation, and blood samples were taken for a complete blood count (CBC), total IgE, and eosinophil isolation. Additionally, a BAC using house dust mite allergen (D. pteronyssinus) was administered to all subjects in the AA group, and all clinical and experimental tests were repeated 24 hours later.

Complete Blood Count and Serum Total Immunoglobulin E

During the investigated subjects’ clinical examination blood samples were collected and delivered to the laboratory in the Department of Laboratory Medicine, LUHS Hospital Kaunas Clinics. The immunoassay analyzer AIA-2000 (Tosoh Bioscience, South San Francisco, CA, USA) was used to measure serum total immunoglobulin (Ig) E concentration. The hematology analyzer UniCel^® DxH 800 Coulter^® Cellular Analysis System (Beckman Coulter, Miami, FL, USA) was used for a complete blood count test.

Lung Function Testing

During the screening and experimental visits all investigated individuals underwent spirometry testing with an ultrasonic spirometer (Ganshorn Medizin Electronic, Niederlauer, Germany). Each individuals’ gender, age, and body height were recorded to later adjust the results according to the predicted values derived from the standard methodology. Then the measured results for forced vital capacity (FVC), forced expiratory volume in 1 second (FEV₁), and the FEV₁/FVC ratio were recorded as the largest of the three independent measurements, as previously described.²³

Airway Hyperresponsiveness Evaluation

The methacholine challenge test was performed to all individuals comprising the AA group to assess airway hyperresponsiveness. Patients were instructed to inhale aerosolized methacholine at 2-minute intervals using a pressure dosimeter (ProvoX, Ganshorn Medizin Electronic, Niederlauer, Germany) with the first administered dose of 0.0101 mg and progressively increasing up to 0.121, 0.511, up to 1.31 mg overall amount was achieved, or until a 20% decrease in FEV₁ from the baseline was measured. The bronchoconstriction effect of each methacholine dose was expressed as previously described.²³

Bronchial Allergen Challenge Test

The bronchial allergen test with inhaled D. pteronyssinus allergen (DIATER, Madrid, Spain) was done on all individuals from the AA group. The examined patient was tasked to inhale the allergen at 10-minute intervals via pressure dosimeter (ProvoX, Ganshorn Medizin Electronic, Niederlauer, Germany). The procedure was interrupted once FEV₁ decreased by ≥20%, as previously described.²³

Skin Prick Testing

Skin prick testing was done to all investigated subjects with standardized allergen extracts (Stallergenes, S.A., Antony, France) for D. pteronyssinus and other clinically relevant allergens. A saline solution was used as a negative control, while a 1% histamine dihydrochloride solution was used as a positive control. The skin prick test results were interpreted following 15 minutes post-application. A >3 mm wheel diameter was interpreted as a positive result reaction to the allergen.

Fractional Exhaled Nitric Oxide Measurement

All study participants’ FeNO was measured by a compact Vivatmo-me device (Bosch Healthcare Solutions, Waiblingen, Germany) according to the manual. During the experimental visit, following a short device self-calibration, the study participant was instructed to carefully and steadily exhale through the mouthpiece for 5 s, and the procedure was done twice. To prevent cross-contamination, personnel used protective equipment, the device was sanitized between uses, and the mouthpiece was used once and then discarded after each study subject.

Peripheral Blood Eosinophil Isolation, Purification, and Subtyping

Peripheral blood samples (up to 30mL of whole blood) were collected in sterile vacutainers with ethylenediaminetetraacetic acid (EDTA) (BD Vacutainer Systems). Granulocyte isolation from whole blood, total eosinophil purification, and subtyping into iEOS-like and rEOS-like cells were performed in a magnetic-activated cell sorting column, as previously characterized.²³ This process utilized magnetic beads conjugated with antibodies against CD62L (L-selectin) (Miltenyi Biotec, Bergisch Gladbach, Germany), a marker expressed mainly by rEOS-like cells, though not on iEOS-like cells.^24,25 After separation, both cell populations were confirmed using a flow cytometer, FACS Calibur (BD Biosciences, San Jose, CA, USA). Additionally, isolated eosinophils were counted, and their viability was assessed using an automated cell counter ADAM (NanoEnTek Inc., Mountain View, CA, USA). The isolated peripheral blood eosinophil sample was used after matching the purity (>96%) and viability (≥95%) criteria. All data presented in the manuscript were from investigated subjects who met these criteria. Additionally, flow cytometry graphs depicting purified ex vivo blood eosinophils (depicted in Figure 2a) and subsequent blood eosinophil subtypes labeled with CD62L antibodies are presented in Figure 2b and c.

Figure 2.

Purified blood eosinophils. (a) Dot plot of all blood eosinophils labeled with CD62L-FITC antibody, excluding cell debris (with forward scatter/side scatter), and gated using a fixed threshold. (b) Fluorescence histogram of all blood eosinophils labeled with CD62L-FITC antibody. (c) Merged fluorescence histograms of all blood eosinophils without CD62L-FITC antibodies; iEOS-like and rEOS-like cells labeled with CD62L-FITC antibodies.

Abbreviations: aEOS, all eosinophils; CD62L, L-selectin; iEOS, inflammatory eosinophil; FITC, Fluorescein isothiocyanate; rEOS, resident eosinophil; SSC, side scatter.

Blood Eosinophil Subtypes ROS Production Evaluation

ROS production in eosinophils was evaluated using a non-toxic DHR-123 fluorescent probe dye (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany). After isolating the investigated subjects’ iEOS and rEOS, experimental flow cytometry tubes (Corning Falcon, Newport, Tennessee, USA) were filled with 5×10⁴ of a blood eosinophil subtype, supplemented with recombinant human IL-3, IL-5, or GM-CSF (10 ng/mL) (Gibco by Life Technologies, Paisley, UK) and PBS to a final volume of 0.2 mL. Each cytometry tube with eosinophils was resuspended and incubated for 90 min at 5% CO₂ 37 °C. An additional tube with eosinophils and PBS, without cytokines, was incubated to calibrate the flow cytometer. Following incubation, each tube was supplemented with freshly prepared DHR-123 (750 ng/mL v/v), resuspended, and incubated for 45 minutes, an adequate duration³⁶ for investigating intracellular ROS production, at 5% CO2 37 °C. After incubation, the spontaneous ROS production was assessed using a flow cytometer by measuring mean fluorescence intensity in the eosinophil subtype population.

Blood Eosinophil Subtypes Chemotaxis Evaluation

Eosinophil chemotaxis was evaluated using transwell inserts (Nalge Nunc International, Rochester, NY, USA) with a semi-permeable membrane and 8 µm-sized pores. First, the 24-well experimental plate was filled with Dulbecco’s modified Eagle’s medium (DMEM) (1% fetal bovine serum (FBS), v/v), supplemented with IL-3, IL-5, GM-CSF (20 ng/mL), or eotaxin-1, 2, 3, and RANTES (100 ng/mL) (Gibco). A carrier with inserts was then gently placed into the plate, avoiding air bubbles beneath the inserts. Each upper insert was supplemented with DMEM without FBS and 2.5×10⁴ of the investigated cell subtype. The plate was incubated under standard conditions for 90 minutes, providing sufficient time for eosinophilopoietins to diffuse to the upper layer, thereby forming a gradient across the membrane and facilitating eosinophil chemotaxis. Following incubation, each lower insert well was supplemented with a 5 mM EDTA solution and incubated for up to 3 minutes. The plate was then gently tapped to detach any eosinophils adhering to the lower side of the insert pore membrane. Next, the bottom well medium was collected into sterile Eppendorf^® tubes and centrifuged at 500 × g for 10 minutes. After centrifugation, the supernatant was discarded, and the cell pellets were resuspended in fresh DMEM solution, transferred to flow cytometry tubes (Corning Falcon), and quantified using a flow cytometer. The percentage of migrated eosinophil subtype was calculated from the total number of viable cells added to each well.

Blood Eosinophil Subtypes Adhesion Evaluation

In preparation for each study subject’s visit, healthy human ASMC were seeded (5 × 10³ viable cells in each well) into 24-well cell culture-treated plates (CytoOne, StarLab, Brussels, Belgium) and cultured in previously described conditions.²³ The ASMC culture line was sourced from a single healthy donor and immortalized by stable expression of human telomerase reverse transcriptase as described by Gosens et al.³⁷ To avoid diminishing cell activity and viability after a repeated number of cell passages, novel cells of the mainline were thawed each time after 6 passages. Once blood eosinophil subtypes were isolated and their viability assessed, co-cultures with ASMC were prepared by adding 1.25×10⁴ of an eosinophil subtype to each well and supplementing with either IL-3, IL-5, or GM-CSF (10 ng/mL). ASMC confluence at assay was approximately 4 × 10⁴, and the eosinophil ratio amounted to 1:3 for each ASMC. Eosinophil adhesion to ASMC after 1 h of incubation was measured by eosinophil peroxidase (EPX) assay as previously described.²³ In addition, blood iEOS-like and rEOS-like cells’ EPX linearity absorbance graphs are provided in Figure 3. Results were quantified as a percentage of the attached eosinophils from the total amount added, derived from the control eosinophil subtype absorbance values (without cytokines).

Figure 3.

Blood eosinophil subtypes adhesion assay. (a) Blood iEOS-like cells EPX absorbance.(b) Blood rEOS-like cells EPX absorbance.

Abbreviations: EPX, eosinophil peroxidase; iEOS, inflammatory eosinophil; rEOS, resident eosinophil.

Statistical Analysis

Statistical data analysis was conducted using GraphPad Prism 8 for Windows (ver. 8.0.1, 2018; GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). Data distribution normality was assessed with the Shapiro–Wilk test. The data distribution did not meet the normality criterion; therefore, non-parametric tests were applied. Differences between two independent groups were evaluated using the Mann–Whitney two-sided U-test. The Wilcoxon matched-pairs signed-rank two-sided test was utilized for two dependent groups. A p < 0.05 value was deemed statistically significant.

Results

Study Subject Characteristics

This study included 13 steroid-free, non-severe allergic asthma (AA) patients, 12 severe eosinophilic asthma (SEA) patients, and 10 healthy control subjects (HS). Table 2 presents the demographic and clinical characteristics of the investigated subjects. All AA group patients were allergic to D. pteronyssinus house dust mites. AA and SEA patients had significantly higher peripheral blood eosinophil count, FeNO, and total IgE, p < 0.05, compared to the HS group. Furthermore, asthma patients exhibited worse lung function than the HS group.

Table 2.

Demographic and Clinical Characteristics of the Study Population

	AA Patients		SEA Patients	Healthy Subjects
Number, n	13		12	10
Sex, male/female, n	6/7		4/8	4/6
Age, median (range), years	25 (18–51)		59 (45–69)##,§	26.5 (23–43)
BMI, median (range), kg/m2	22.2 (18.9–40.6)		27.2 (17.6–40.8)#,§	22.75 (19.5–31.4)
Sensitization to D. pteronyssinus, n	13		2	0
PD20m, geometric mean (range), mg	0.196 (0.070–0.675)		ND	ND
PD20a, geometric mean (range), HEP/mL	2.138 (1–20)		ND	ND
	Baseline	24 h after BAC
FEV1, L	3.27 ± 0.17#	3.06 ± 0.23	1.64 ± 0.144##,§	3.92 ± 0.259
FEV1, % of predicted	84.3 ± 3.7#	75.5 ± 4.2*	63.7 ± 6.7##,§	96.8 ± 2.5
Blood eosinophil count, (×109)/L	0.362 ± 0.064##	0.492 ±0.076*	0.892 ± 0.303##,§	0.110 ± 0.010
Blood eosinophil count, %	6.09 ± 1.03##	8.38 ± 1.54*	7.80 ± 1.50	2.15 ± 0.28
IgE, IU/mL	637.8 ± 246.6##	630.8 ± 248.7	219.2 ± 122.5#	21.0 ± 5.2
FeNO, ppb	52.3 ± 9.2##	71.5 ± 12.2	40.3 ± 6.0##	12.1 ± 2.5

Notes: ^#p < 0.05, ^##p < 0.01 compared with the healthy subject group; ^§p < 0.05, compared with the AA group; *p < 0.05, compared within the AA group before BAC. Data presented as mean ± standard error of the mean, or as stated within the row.

Abbreviations: AA, allergic asthma; BAC, bronchial allergen challenge; BMI, body mass index; D. Pteronyssinus, Dermatophagoides pteronyssinus; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in one second; IgE, immunoglobulin E; ND, not done; SEA, severe eosinophilic asthma; PD20_m, provocation dose of methacholine resulting in a 20% decrease in FEV₁; PD20_a, provocation dose of allergen resulting in a 20% decrease in FEV₁.

Comparing asthma phenotypes, SEA patients had significantly lower FEV₁ and higher absolute blood eosinophil count, while AA patients had elevated total serum IgE levels. AA patients’ FEV₁ (% of predicted) significantly decreased, while the blood eosinophil count and FeNO increased 24 h after BAC, p < 0.05, with no changes in total IgE.

Eosinophilopoietins Effect on Blood Eosinophil Subtypes Spontaneous ROS Production

During chronic airway inflammation, eosinophils are also activated by various mediators, leading to increased spontaneous ROS production. This may clinically contribute to bronchial hyperreactivity and airway inflammation.³⁸ In addition, we previously demonstrated that blood eosinophil spontaneous ROS production at baseline may vary between asthma patients and healthy individuals,³⁹ while in this study we further investigated the effects of IL-3, IL-5, and GM-CSF on the spontaneous production of ROS in blood eosinophil subtypes.

AA patients’ blood iEOS-like cells, compared to rEOS-like cells, exhibited higher spontaneous ROS production at baseline, p < 0.05, and following incubation with IL-3, IL-5, or GM-CSF, p < 0.05. (Figure 4a). In the SEA group, no differences were observed between blood eosinophil subtypes. However, the spontaneous ROS production of SEA patients’ rEOS-like cells was significantly enhanced at baseline and after incubation with IL-3, IL-5, and GM-CSF, p < 0.05, compared to AA patients’ rEOS-like cells. This trend in iEOS-like cells was only observed following incubation with IL-5 and GM-CSF, p < 0.05.

Figure 4.

Blood eosinophil subtypes’ spontaneous ROS production. (a) All investigated groups after incubation with eosinophilopoietins. (b) Allergic asthma group at baseline and 24 h after bronchial allergen challenge.

Notes: Allergic asthma n=9. Severe eosinophilic asthma n=7. Healthy subjects n=7. Dots represent individual donors. # p < 0.05, ## p < 0.01, ### p < 0.001 - compared with the respective blood eosinophil subtype of the healthy subject group. * p < 0.05, ** p < 0.01, - compared with the respective iEOS-like cells column within the AA group. &p < 0.05, &&p < 0.01, &&&p < 0.001 - compared with the respective blood eosinophil subtype of the AA group. Results are shown as mean ± standard error of the mean. P-values shown; no false discovery rate correction applied. Differences between two investigated subject groups; or between eosinophil subtypes were evaluated using the Mann–Whitney two-sided U-test. The Wilcoxon matched-pairs signed-rank two-sided test was used for eosinophil subtype comparisons within the same study group following activation with cytokines.

Abbreviations: iEOS, inflammatory eosinophil; GM-CSF, granulocyte macrophage-colony stimulating factor; rEOS, resident eosinophil; IL, interleukin; V1, first experimental visit (baseline); V2, second experimental visit (24 h after bronchial allergen challenge).

Additionally, the spontaneous ROS production of blood iEOS-like cells from AA and SEA patients increased after incubation with IL-3, IL-5, and GM-CSF, p < 0.05, compared to baseline iEOS-like cells. However, the ROS production of rEOS-like cells was affected unevenly. In the AA group, the ROS production of rEOS-like cells increased after incubation with IL-3 and IL-5. In the SEA group, only IL-5 and GM-CSF significantly influenced the rEOS-like cells, p < 0.05. Spontaneous ROS production was significantly enhanced in blood eosinophil subtypes from both asthma groups, p < 0.05, compared to the HS group. Meanwhile, in the HS group, only GM-CSF significantly promoted ROS production in iEOS-like cells, p = 0.0313.

After evaluating differences between asthma phenotypes, it was determined that the spontaneous ROS production of SEA patients’ rEOS-like cells was significantly enhanced at baseline and after incubation with IL-3, IL-5, and GM-CSF, p < 0.05, compared to AA patients’ rEOS-like cells. This trend in iEOS-like cells was only observed following incubation with IL-5 and GM-CSF, p < 0.05.

Additionally, we investigated the spontaneous ROS production in blood eosinophil subtypes from AA patients at baseline and 24 hours after BAC with D. Pteronyssinus (Figure 4b). The ROS production of blood eosinophil subtypes was significantly enhanced following BAC, p < 0.05. Incubating allergen-activated blood iEOS-like and rEOS-like cells with IL-3, IL-5, and GM-CSF significantly increased spontaneous ROS production, p < 0.05, compared to eosinophilopoietins’ effect on blood eosinophils isolated before BAC. The ROS production from allergen-activated iEOS-like cells after incubation with IL-3, IL-5, or GM-CSF remained significantly higher than that of rEOS-like cells, p < 0.05. Additional data provided in Supplementary Table 1.

Eosinophilopoietins and C-C Chemokines Effect on Chemotaxis of Blood Eosinophil Subtypes

In response to type 2 cytokines and C-C chemokines produced by lung structural cells and Th2, ILC2 cells at the inflammatory site, eosinophils accumulate in the airways and contribute to the development of asthma.⁴⁰ Thus, we investigated the chemotaxis of blood eosinophil subtypes towards eosinophilopoietins (IL-3, IL-5, GM-CSF) and C-C chemokines (eotaxin-1, eotaxin-2, eotaxin-3, and RANTES) in asthma.

We found that the blood eosinophil subtypes of AA and SEA patients exhibited significantly enhanced chemotaxis at baseline and in response to eosinophilopoietins, p < 0.05, compared to the HS group (Figure 5a). In AA patients, the chemotaxis of iEOS-like cells towards IL-3, IL-5, and particularly GM-CSF was higher, p < 0.05, compared to control iEOS-like cells without incubation with eosinophilopoietins. Similarly, SEA patients’ iEOS-like cells showed increased chemotaxis towards IL-3, IL-5, and GM-CSF, p < 0.05. In the HS group, a small yet significant chemotactic response of iEOS-like cells only towards IL-3 was noted, p < 0.01. Meanwhile, rEOS-like cells, isolated from all investigated groups, exhibited a positive chemotactic response to eosinophilopoietins; however, no significant changes compared to baseline were found.

Figure 5.

Blood eosinophil subtypes chemotaxis. (a) All investigated groups after incubation with eosinophilopoietins. (b) Allergic asthma group at baseline and 24 h after bronchial allergen challenge.

Notes: Allergic asthma n=9. Severe eosinophilic asthma n=9. Healthy subjects n=9. Dots represent individual donors. # p < 0.05, ## p < 0.01, ### p < 0.001 - compared with the respective blood eosinophil subtype of the healthy subject group. * p < 0.05, compared with the respective rEOS-like cells column. Results are shown as mean ± standard error of the mean. P-values shown; no false discovery rate correction applied. Differences between two investigated subject groups; or between eosinophil subtypes were evaluated using the Mann–Whitney two-sided U-test. The Wilcoxon matched-pairs signed-rank two-sided test was used for eosinophil subtype comparisons within the same study group following activation with cytokines.

Abbreviations: iEOS, inflammatory eosinophil; GM-CSF, granulocyte macrophage-colony stimulating factor; rEOS, resident eosinophil; IL, interleukin; V1, first experimental visit (baseline); V2, second experimental visit (24 h after bronchial allergen challenge).

The chemotaxis of blood iEOS-like cells in AA patients 24 hours after BAC was significantly enhanced, compared to non-allergen-activated iEOS-like cells, p < 0.05. (Figure 5b). In addition, allergen-activated iEOS-like and rEOS-like cells displayed a significant chemotactic response to IL-5, p < 0.05, and only iEOS-like cells reacted to IL-3, compared with respective cell subtype chemotaxis before BAC. Additional data provided in Supplementary Table 2.

After investigating the effect of C-C chemokines on blood eosinophil subtypes’ chemotaxis, we found that blood iEOS-like cells in all investigated groups, while rEOS-like cells only in asthma groups, exhibited an increased chemotactic response to eotaxin-1, eotaxin-2, eotaxin-3, and RANTES, p < 0.05 (Figure 6a). The chemotaxis of AA patients’ blood iEOS-like and rEOS-like cells towards eotaxin-3, as well as rEOS-like cells towards RANTES, was enhanced when compared with the HS group, p < 0.05. Meanwhile, the chemotaxis of SEA patients’ iEOS-like cells towards eotaxin-3 and RANTES, and the chemotaxis of rEOS-like cells to all investigated chemokines, were significantly higher compared to the HS group, p < 0.05. Among the chemokines investigated, eotaxin-3 had a greater effect on the chemotaxis of AA patients’ iEOS-like cells, p < 0.05, compared to eotaxin-1, eotaxin-2, and RANTES.

Figure 6.

Blood eosinophil subtypes chemotaxis. (a) All investigated groups after incubation with C-C chemokines. (b) Allergic asthma group at baseline and 24 h after bronchial allergen challenge.

Notes: Allergic asthma n=8. Severe eosinophilic asthma n=8. Healthy subjects n=7. Dots represent individual donors. # p < 0.05, ## p < 0.01, ### p < 0.001 - compared with the respective blood eosinophil subtype of the HS group. &p < 0.05, &&p < 0.01 - compared with the respective blood eosinophil subtype column of the AA group. Results are shown as mean ± standard error of the mean. P-values shown; no false discovery rate correction applied. Differences between two investigated subject groups; or between eosinophil subtypes were evaluated using the Mann–Whitney two-sided U-test. The Wilcoxon matched-pairs signed-rank two-sided test was used for eosinophil subtype comparisons within the same study group following activation with chemokines.

Abbreviations: iEOS, inflammatory eosinophil; RANTES, regulated on activation, normal T cell expressed and secreted; rEOS, resident eosinophil; V1, first experimental visit (baseline); V2, second experimental visit (24 h after bronchial allergen challenge).

We also compared the chemotaxis of blood eosinophil subtypes from AA patients towards eotaxin-1, −2, −3, and RANTES before BAC and 24 hours later (Figure 6b). Among the chemokines investigated, the chemotaxis of iEOS-like cells to eotaxin-3 was significantly higher compared to iEOS-like cells before BAC. Furthermore, the response of iEOS-like cells to eotaxin-3 was greater than that of rEOS-like, p < 0.05. Similar trends were observed with other chemokines; however, no significant changes were found. Additional data provided in Supplementary Table 3.

Eosinophilopoietins Effect on Blood Eosinophil Subtypes’ Adhesion to Airway Smooth Muscle Cells

Hypothesizing that different eosinophil subtypes may play a role in airway remodeling through accumulation and direct adhesion to lung structural cells, we compared the effects of IL-3, IL-5, and GM-CSF on the adhesion of blood eosinophil subtypes to ASMC.

At baseline, AA and SEA patients’ iEOS-like and rEOS-like cell adhesion to ASMC was higher than in the HS group, p < 0.05 (Figure 7a). IL-3, IL-5, and GM-CSF enhanced blood iEOS-like cell adhesion to ASMC in AA and SEA patients, p < 0.05, compared to baseline iEOS-like cell adhesion. Meanwhile, rEOS-like cell adhesion significantly increased in both asthma groups solely with GM-CSF, p < 0.01.

Figure 7.

Eosinophil adhesion to airway smooth muscle cells. (a) All investigated groups after incubation with eosinophilopoietins. (b) Allergic asthma group at baseline and 24 h after bronchial allergen challenge.

Notes: Allergic asthma n=9. Severe eosinophilic asthma n=9. Healthy subjects n=9. Dots represent individual donors. Results are shown as mean ± SEM. # p < 0.05, ## p < 0.01, ### p < 0.001 - compared with the respective blood eosinophil subtype of the HS group. &p < 0.05 - compared with the respective blood eosinophil subtype of the AA group. Results are shown as mean ± standard error of the mean. P-values shown; no false discovery rate correction applied. Differences between two investigated subject groups; or between eosinophil subtypes were evaluated using the Mann–Whitney two-sided U-test. The Wilcoxon matched-pairs signed-rank two-sided test was used for eosinophil subtype comparisons within the same study group following activation with cytokines.

Abbreviations: iEOS, inflammatory eosinophil; GM-CSF, granulocyte macrophage-colony stimulating factor; rEOS, resident eosinophil; IL, interleukin; V1, first experimental visit (baseline); V2, second experimental visit (24 h after bronchial allergen challenge).

In the HS group, only GM-CSF significantly enhanced iEOS-like and rEOS-like cells adhesion to ASMC, p < 0.05. Furthermore, rEOS-like cell adhesion was greater than that of iEOS-like cells, p < 0.05. Following the BAC in AA patients, the adhesion of isolated blood iEOS-like cells to ASMC was significantly increased, p < 0.05, compared to their adhesion before BAC (Figure 7b). Furthermore, both blood eosinophil subtypes’ attachment to ASMC was increased by IL-5, while IL-3 only promoted iEOS-like cells’ adhesion, p < 0.05. GM-CSF had no additional significant impact on either eosinophil subtype. Additional data provided in Supplementary Table 4.

Discussion

This study found that IL-3, IL-5, and GM-CSF upregulate the chemotaxis of iEOS-like cells, spontaneous ROS production, and adhesion to ASMC, with a stronger effect in asthma compared to HS. Meanwhile, these cytokines affect rEOS-like cells differently: IL-5 upregulates ROS production in both asthma groups, while IL-3 only influences rEOS-like cells in the AA group and GM-CSF in the SEA group. SEA patients’ rEOS-like cells’ ROS production following activation with eosinophilopoietins was significantly higher than AA group. The observed eosinophilopoietin effects on blood eosinophil subtypes biological activity are significantly higher in AA and SEA compared to HS. Additionally, eosinophilopoietins show no significant effect on rEOS-like cell chemotaxis, and only GM-CSF promotes rEOS-like cell adhesion across all investigated groups. C-C chemokines eotaxin-1, eotaxin-2, eotaxin-3, and RANTES enhance the chemotaxis of both blood eosinophil subtypes in AA and SEA patients, with eotaxin-3 having a greater impact on iEOS-like cells in AA. Lastly, in vivo allergen-activated blood eosinophil subtype ROS production in AA patients increases further after activation by IL-3, IL-5, and GM-CSF. Eotaxin-3 promotes the chemotaxis of iEOS-like cells, IL-3 influences the chemotaxis and adhesion of iEOS-like cells, and IL-5 upregulates the adhesion of both eosinophil subtypes.

Asthma pathogenesis can be driven by various inflammatory or structural lung cells and their secreted mediators. Eosinophils have become the primary focus of biological therapy for treating severe asthma patients with blood and/or airway eosinophilia.⁴¹ Monoclonal antibodies, specifically benralizumab, reslizumab, and mepolizumab, target the eosinophilic inflammation pathway - IL-5 and IL-5 receptor.⁴² In this study, blood eosinophil attachment to ASMC was inferred from linear EPX activity, as shown in Figure 3; however, complementary direct counts via flow cytometry or hemocytometer could further corroborate the results. Still, our study shows that GM-CSF significantly affects blood eosinophil subtypes, particularly rEOS-like cells’ attachment to ASMC and the chemotaxis of iEOS-like cells, indicating that GM-CSF can act directly as a chemoattractant, in addition to known enhancement of eosinophil survival, activation, and responsiveness to chemokines.^5,43 Therefore, further dose-dependent studies of GM-CSF are required to determine whether blood eosinophil subtype functions correlate with real-world outcomes in asthma exacerbations, steroid usage, or predicting treatment responsiveness. The previous work of Nobs et al supports these results, suggesting that GM-CSF signaling promotes lung eosinophilia during allergic inflammation and may facilitate eosinophil accumulation in the lung.⁴⁴ Currently, GM-CSF serves as both a tool and a treatment target in various therapeutic practices, including cancer, autoimmune diseases, and sepsis-related immune suppression.⁴⁵ Anti-GM-CSF monoclonal antibody treatment has shown mild success in a clinical setting, demonstrating FEV₁ improvement in eosinophilic asthma after 24 weeks, but with no enhancement in asthma control and exacerbation count during a Phase 2 trial.⁴⁶ This suggests that further asthma phenotyping based on blood and/or tissue eosinophil subtype distribution may be necessary to achieve a significant treatment response. After thoroughly assessing the patient’s condition and identifying the underlying disease phenotype, GM-CSF could potentially become a therapeutic target for treating eosinophilia-driven diseases.

The main symptom of eosinophilic inflammation and asthma is the infiltration of eosinophils in areas of allergen exposure in atopic individuals, where they degranulate and release intracellular mediators - granule proteins, cytokines, and chemokines.^5,47 For the eosinophil to migrate to the site of inflammation, it must adhere to vascular endothelial cells; this interaction is mediated by the α4 integrin and VCAM-1.⁴⁸ After eosinophil adhesion to endothelial cells, C-C chemokines such as eotaxin or RANTES stimulate migration across endothelial cells expressing VCAM-1. Additionally, activated Th2, ILC2, and airway epithelial cells secrete various mediators, including IL-3, IL-5, and GM-CSF, which may activate eosinophils and act as potential chemoattractants for iEOS-like cells. It has been found that blood eosinophils isolated from the peripheral blood of patients with allergic rhinitis and mild atopic asthma displayed a positive, but non-significant, chemotactic response to IL-3 and IL-5 (experimental concentration 10 mg/mL).⁴⁹ Additionally, eotaxin-1, eotaxin-2, and particularly eotaxin-3, induced the migration of peripheral blood eosinophils from healthy individuals and asthma patients.⁵⁰ During our study, blood eosinophils were first divided into iEOS-like and rEOS-like cells, showing that the blood iEOS-like cell chemotaxis in AA and SEA patients significantly increased in response to all the cytokines and chemokines, compared to baseline. Meanwhile, no significant chemotactic response of rEOS-like cells to IL-3, IL-5, or GM-CSF was observed in any of the groups studied, and C-C chemokines had a non-significant effect on the chemotaxis of rEOS-like cells in healthy subjects. Previously, an increase in CCR3 expression has been found in CD62^low blood iEOS cells.²² During this study we found that all eotaxins, especially eotaxin-3, promoted the chemotaxis of allergen-activated iEOS-like cells, indicating that it is the potentially key chemokine for eosinophil migration into inflamed airways. In response to various mediators, iEOS-like cells, considered negative due to their correlation with worse asthma control test scores and exacerbation history,³² may accumulate in sites of inflammation and harm the surrounding tissues.

Eosinophils exert their functions by secreting various mediators and may also produce ROS, thus contributing to local tissue oxidative stress. Inflammatory eosinophils are primarily involved in the immune response and migrate closer to the airway epithelium due to the production of alarmins (thymic stromal lymphopoietin, IL-25, and IL-33) and type 2 cytokines by Th2, ILC2, and epithelial cells following irritation by parasites, fungi, allergens, or viruses.⁵¹ Jones et al evaluated the total ROS production of eosinophils after stimulation with IL-3, IL-5, or GM-CSF (10 ng/mL), and found that all three cytokines stimulated the ROS production in eosinophils.⁵² We extended this study by examining the characteristics of spontaneous ROS production in blood eosinophil subtypes. Throughout the study, the trends remained similar: IL-3, IL-5, and GM-CSF stimulated spontaneous ROS production in eosinophil subtypes of AA and SEA patients; however, the ROS production of iEOS-like cells was dominant in the AA group. In addition, the SEA group rEOS-like cell ROS production was the highest among all the groups investigated. Lastly, after BAC, the spontaneous ROS production of allergen-activated eosinophil subtypes was further enhanced, suggesting that allergen exposure in the airways triggers local dendritic, Th2, and epithelial cells to release T2 cytokines⁵³ which prime innate immune cells, including eosinophils. In addition, iEOS-like cells remained the dominant ROS-producing cell type, which supports their role as the pro-inflammatory eosinophil subtype compared to rEOS-like cells. Various studies have shown that serum levels of IL-3, IL-5, GM-CSF, and eotaxin were significantly higher in patients with asthma and often correlated with the severity of the disease.^54–57 Due to the increased concentration of chemokines and cytokines, iEOS-like cells that first migrate to the epithelial area become further activated, potentially producing more ROS and contributing to respiratory tract damage caused by oxidative stress. Exposure to environmental agonists has been shown to induce excess ROS production, resulting in the disruption of normal physiological functions of DNA, proteins, and lipids, which clinically manifest as increased bronchial reactivity and inflammation.⁵⁸ Results from our study indicate that iEOS-like cell co-activation by allergens and eosinophilopoietins may lead to excessive ROS production and cytotoxic granule protein release, promoting airway inflammation in patients with AA.

For this study, the 24-hour time point after BAC was selected to correspond to the characterized late-phase allergic response, during which eosinophil recruitment peaks and coincides with maximal Th2 cytokine and chemokine activity. The 24-hour window is informative for eosinophil trafficking and activation because it captures the eosinophil activation (via chemokines, IL-5/IL-13 signaling and adhesion molecule upregulation), therefore reflecting the key mechanistic link between allergen exposure and the sustained airway characteristics of asthma.^34,35 Nonetheless, additional timepoints of investigation during the 12–24 h window and up to 72 h from sputum and/or lung samples could provide valuable insights into whether eosinophil subtypes change activation states, sustain ROS production or degranulation, and persist in the inflamed airways. Due to eosinophils being associated with the late-phase reaction, sampling bone marrow following the early-phase reaction after BAC could uncover early transcriptional or cytokine-driven signaling pathways that lead to priming of eosinophil progenitor cells to develop into distinct mature cell subtypes. In addition, during this study, ex vivo blood eosinophils were separated based on surface CD62L. However, based on the existing literature, additional blood eosinophil subtyping based on surface IL-3R and/or IL-101 could provide valuable insights into the functional dichotomy of iEOS-like and rEOS-like cells in asthma. GM-CSF and eotaxin-3 revealed to be the most effective at upregulating blood eosinophil subtype adhesion and chemotaxis; therefore, future studies should include broader dose-response analysis to clarify threshold effects that may have been obscured by the limited concentration range in this study. Moreover, increasing the sample size and adjusting study models to include age and ICS exposure would clarify age and treatment-related variability in blood eosinophil activity and response to cytokines.

A potential limitation of our eosinophil subtype study model is that after completing the purification of blood eosinophils from granulocytes, the rEOS-like (CD62L⁺) cells were separated using positive selection with magnetic beads, while the unselected cells were identified as iEOS-like (CD62L⁻) cells. Recent blood eosinophil studies differentiate blood iEOS as CD62L^low and rEOS as CD62L^bright cells.²² Meanwhile, regarding the MACS separation method used in this study, the low level of potentially expressed CD62L, as shown in Figure 2, is inadequate for suspension in the magnetic field column; therefore, only a small portion of iEOS-like cells could theoretically be captured in the magnetic separation column. Another potential limitation is that only a single-dose stimulus of eosinophils was used. This study was designed to evaluate the qualitative and comparative effects of eosinophilopoietins and C-C chemokines on blood eosinophil subtypes based on previously published data that have shown robust or pathophysiologically relevant activation of eosinophils. The authors acknowledge that a full dose-response analysis could offer additional quantitative insights; however, the chosen single-dose comparisons are appropriate for evaluating relative biological cytokine effects on ex vivo blood eosinophils. Furthermore, the sample sizes in this study were small, and nominal p-values were reported without additional multiple-testing adjustments such as false discovery rate as it could further reduce the ability to detect true group differences when the potential effect sizes are modest. Still, the participants were thoroughly investigated before admission into the study as described in the inclusion and exclusion criteria section to reduce variability and enable more precise interpretation of the studied effects. Moreover, our findings were thoroughly discussed and compared with previously published data and biological disease mechanisms, providing additional validity to the results. In addition, a potential limitation regarding BAC follow-up is that only a single time point was chosen to repeat the experiments. Nonetheless, the 24 h follow-up was selected based on previous studies demonstrating that it is the peak of the late-phase allergic response and eosinophil activation following an allergen attack.³³ The authors acknowledge that additional time points could contribute to a more detailed profiling of early-intermediate or late phase changes in airway inflammatory responses; however, the 24 h follow-up remains an informative and established window for assessing eosinophil activation and changes in biological response to cytokine stimuli. Lastly, a potential limitation of this study is that SEA patients were significantly older than AA patients and HS, and were receiving high doses of inhaled steroids, which may have affected eosinophil activity. Mathur et al have previously revealed that no differences in blood eosinophil chemotactic and adhesive properties were found between the older and younger asthma patient groups (55–80 vs 20–40 years of age), while eosinophil stimulation with phorbol myristate acetate suggested a non-significant decrease of superoxide anion production in the older asthma group.⁵⁹ It is known that inhaled steroids can switch off the inflammatory cascade through inhibition of pro-inflammatory transcription factors.^60,61 This may lead to reduced eosinophil migration, transepithelial passage, and recruitment to the airways^62,63 or decreased integrin-mediated adhesion to lung structural cells.^64,65 Inhaled steroid therapy may also suppress superoxide production in a dose-dependent manner, although effects on H₂O₂ down-regulation are minimal.^66,67 Therefore, we acknowledge potential bias in the eosinophil activity data in the SEA group due to the effect of inhaled steroids.

Conclusion

Eosinophilopoietins and C-C chemokines increase chemotaxis, adhesion to ASMC, and ROS production of ex vivo blood eosinophil subtypes, especially iEOS-like cells, with these effects being more significant in adult asthma patients. The co-activation of iEOS-like cells by allergens and eosinophilopoietins may promote excessive spontaneous ROS production in allergic asthma. The results from this study suggest mechanistic insights that GM-CSF and eotaxin-3/CCL26 are the most potent promoters of eosinophil chemotaxis and adhesion, particularly of iEOS-like cells. Nonetheless, our findings are exploratory and considering that GM-CSF receptor and CCR3 lack eosinophil specificity, it is essential to conduct more extensive studies to establish these cytokines in the current landscape of biologic therapies for asthma and eosinophilia-driven diseases.

Funding Statement

This research was funded by the Science Foundation of the Lithuanian University of Health Sciences.

Abbreviations

AA, allergic asthma; ASMC, airway smooth muscle cells; BAC, bronchial allergen challenge; CBC, - complete blood count; CCL, C-C motif ligand; CCR, C-C motif chemokine receptor; ; DHR, dihydrorhodamine; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic; EPX, eosinophil peroxidase; FBS, fetal bovine serum; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; GM-CSF, granulocyte macrophage-colony stimulating factor; HS, healthy subject; iEOS - inflammatory eosinophil; IgE, immunoglobulin E; IL, interleukin; ILC, innate lymphoid cell; LUHS, Lithuanian University of Health Sciences; PBS, phosphate-buffered saline; RANTES, regulated on activation, normal T cell expressed and secreted; rEOS, resident eosinophil; ROS, reactive oxygen species; SEA, severe eosinophilic asthma; SEM, standard error of the mean; Th2, T helper type 2 cells; V1, first experimental visit; V2, second experimental visit - 24 h after bronchial allergen challenge.

Data Sharing Statement

The datasets analyzed and obtained in this study are available from the corresponding author upon reasonable request. The manuscript includes all relevant clinical and experimental data of the study participants, and no additional unpublished data from the study will be made available elsewhere. The authors declare no intent to share individual de-identified study participant data.

Author Contributions

Airidas Rimkunas: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Conceptualization

Andrius Januskevicius: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review & editing, Conceptualization

Egle Vasyle: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Conceptualization

Virginija Kalinauskaite-Zukauske: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Conceptualization

Jolita Palacionyte: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Conceptualization

Skaidrius Miliauskas: Data curation, Methodology, Resources, Writing – review & editing, Conceptualization

Kestutis Malakauskas: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing, Conceptualization, Software, Project administration, Funding acquisition

All authors took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report there are no conflicts of interest in this work. The abstract of this paper was presented at the “16th Lithuanian Young Scientists’ conference “Biofuture: Perspectives in Natural and Life Sciences” as a conference talk with interim findings. The abstract was published in “Conference Abstracts” in “16th Lithuanian Young Scientists’ conference” Abstract book, ISSN 2783-826.