Eosinophil-derived neurotoxin levels can predict allergic disease development and atopic march in children

Zak Callaway; Chang-Keun Kim

doi:10.3345/cep.2024.01382

Clin Exp Pediatr > Volume 68(6); 2025

Callaway and Kim: Eosinophil-derived neurotoxin levels can predict allergic disease development and atopic march in children

Review Article

Allergy

Eosinophil-derived neurotoxin levels can predict allergic disease development and atopic march in children

Zak Callaway, PhD, MPHTM^1,²

, Chang-Keun Kim, MD, PhD²

¹Science Division, Mahidol University International College, Mahidol University, Nakhon Pathom, Thailand

²Asthma and Allergy Center, Inje University Sanggye Paik Hospital, Seoul, Korea

Corresponding author: Chang-Keun Kim, MD, PhD. Asthma & Allergy Center, Inje University Sanggye Paik Hospital, 1342 Dongil-ro, Nowon-gu, Seoul 01757, Korea Email: kimck@paik.ac.kr

Received September 10, 2024 Revised December 23, 2024 Accepted December 23, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Clinical and Experimental Pediatrics 2025;68(6):398-405.

Published online: February 3, 2025

DOI: https://doi.org/10.3345/cep.2024.01382

Abstract

In some children, atopic manifestations begin with atopic dermatitis and progress to allergic asthma and allergic rhinitis; of them, a small subset experience food allergies as well. This progression shares genetic and environmental predisposing factors and immunological features, such as allergen-specific T-helper type 2 responses, that manifest as specific immunoglobulin E production and eosinophil activation. Eosinophil-derived neurotoxin (EDN), which is released by eosinophils during this activation, shows promise as a reliable and accurate biomarker. EDN levels are elevated in a subset of patients with atopic march-associated conditions. Elevated EDN levels predict allergic disease development, demonstrating that EDN is a good biomarker for the prognosis, diagnosis, treatment, and monitoring of allergic diseases comprising atopic march. The early measurement of EDN would help identify those who are more likely to develop allergic diseases later in life. Thus, the early detection and treatment of elevated EDN could lead to better outcomes, including halting atopic march.

Key words: Allergic rhinitis, Asthma, Atopic dermatitis, Biomarker, Eosinophil-derived neurotoxin

Key message

· Allergic march occurs in a subset of children, beginning with atopic dermatitis and progressing to food allergies, allergic rhinitis, and/or asthma. Its early diagnosis is important to slowing its progression.

· Eosinophil-derived neurotoxin (EDN), an excellent biomarker of eosinophil activity, is often elevated in allergic diseases.

· EDN levels have been used to predict allergic disease development and diagnose, treat, and monitor allergic diseases.

Graphical abstract. EDN, eosinophil-derived neurotoxin. Data from Terashi et al. Exp Ther Med 2023;26:565 [32].

Introduction

Atopic march refers to the natural history of allergic diseases that develop in infants and children. Typically the "march" begins with atopic dermatitits (AD) and then progresses to immunoglobulin E (IgE)-mediated food allergy (FA), allergic asthma (AA), and allergic rhinitis (AR) (Fig. 1). The progression of allergic conditions shares genetic and environmental predisposing factors with immunologic features, such as one or more allergen-specific T-helper type 2 responses. These responses may manifest as specific IgE production, granulocyte activation (e.g., eosinophils), mucus production, and edema [1]. Each step of atopic march increases an individual's risk of developing allergic conditions.

AD often arises in early childhood; therefore, it is considered the origin of atopic march. However, many allergic conditions are a product of the intricate interplay of genetics, environment, and socioeconomic status with AD, with ethnicity and skin barrier dysfunction as additional risk factors [2]. Some clinicians and researchers caution against overestimating the classical atopic march because it does not consider the heterogeneity of allergic phenotypes [3]. Since AD is often the first step in atopic march, its early diagnosis and swift treatment are seen by many as key opportunities to prevent progression to other allergic diseases. Thus, objective measurement of the underlying immunopathological processes involved in AD, FA, AA, and AR is vital for disease diagnosis, treatment, and monitoring.

Eosinophils are major effector cells in the allergic process; hence, their measurement is invaluable in the treatment and monitoring of eosinophil-related diseases, such as AA, AD, and AR. However, eosinophil numbers/percentages provide minimal information about eosinophil activity. Eosinophil activation leads to the extracellular release of 4 granule proteins: major basic protein, eosinophil peroxidase, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). The focus on reliable and accurate biomarkers of allergic diseases recently shifted from ECP to EDN for several reasons. EDN has superior test reproducibility [4], is more recoverable from test tubes because of its weaker electrical charge, is released with greater efficiency from eosinophils, can be measured in several bodily fluids, and remains stable for more than 1 year when frozen [5]. EDN also has antiviral activity against respiratory pathogens that are strongly associated with allergic disease development, such as respiratory syncytial virus (RSV) and human rhinovirus (hRV) [6].

This review assessed the utility of EDN as a biomarker for the diagnosis, prognosis, treatment, and monitoring of serial allergic conditions, namely AD, FA, AA, and AR, that comprise atopic march.

Atopic march

AD, asthma, AR, and FA are generally considered allergic diseases, although some forms of asthma are nonallergic. Thus, it is necessary to approach diseases such as asthma as heterogeneous in nature to better reflect reality and lead to a more accurate diagnosis and precisely targeted therapy. An early study on atopic march found that patients with AD were at greater risk of developing transient early asthma and persistent but non-allergic late-onset asthma [7]. A more recent study [8] reported quite different disease trajectories in 9,801 children with varying symptoms. Eight distinct disease pathways were identified: no disease (51.3%), atopic march (3.1%), persistent eczema and wheezing (2.7%), persistent eczema with late-onset rhinitis (4.7%), persistent wheezing with late-onset rhinitis (5.7%), transient wheezing (7.7%), eczema (15.3%), and rhinitis (9.6%). This research group concluded that eczema (AD), wheezing, and rhinitis are mostly independent entities, but are all associated with atopic sensitization.

1. Risk factors for atopic march

Multiple studies identified several risk factors associated with atopic march. AD severity and age at onset are factors in the relationship between AD and the development of allergic airway disease; these 2 factors may influence each other. More severe AD is associated with the development of multiple (e.g., asthma and AR) and more persistent diseases, whereas AD severity is the strongest risk factor for early-onset AD [9]. In this study, atopic sensitization was a risk factor for the development of school-age asthma. Another study reported that children with very-early-onset persistent (onset at <6 months of age; still present at 2–5 years of age) and very-early-onset remitting eczema (onset at <6 months of age; absent by 2–5 years of age) had asthma at 18 years of age (odds ratio [OR], 3.2 and 2.7, respectively) [10]. Late-onset eczema (onset at 2–5 years of age) was associated with an increased risk of asthma at 12 years of age (OR, 3.0) but not 6 years of age. Only very-early-onset persistent eczema was associated with an increased risk of AR (OR, 2.4). By analyzing the results of 2 longitudinal birth cohort studies (n=9,894 and n=3,652), Paternoster et al. [11] identified 6 AD subgroups. The risk of asthma development was the highest in the early-onset persistent subgroup at 7 years of age (OR, 5.5) and 13 years of age (OR, 7.9).

Early allergic polysensitization appears to be another risk factor for the development of allergic morbidities such as asthma and AR. Food allergen sensitivity (without aeroallergen sensitization) in the first 24 months of life was associated with a higher risk of asthma in 2 different cohorts (OR, 2.2 and 4.9), with similar results seen for AR. However, when a child was cosensitized to food and aeroallergens, the risk of asthma (OR, 8.3 and 14.4, respectively) and AR (OR, 3.9 and 7.6, respectively) was even higher in each cohort [12]. A meta-analysis performed by the same research group examined data from 13 cohorts and found an association between early food sensitization and an increased risk of wheezing/asthma and AR at 4–8 years of age [13]. The Canadian Health Infant Longitudinal Development Study reported that AD was associated with an increased risk of asthma at 3 years of age only if sensitization to inhalants, foods, or both was present at 1 year of age [14].

A family history of atopy can also increase the risk of the future development of allergic diseases. This suggests that genes may influence the course of atopic march. In the PASTURE (Protection Against Allergy Study in Rural Environments) study [15], a parental history of allergies increased a child's risk of developing AD (OR, 2.87) with an early persistent phenotype (onset <2 years of age) compared to those with no parental history of allergies. Of the 4 AD phenotypes synthesized in this study, 2 were early-onset and associated with FA.

EDN as biomarker for allergic disease

The activity of eosinophils, the major effector cells in allergic processes, is best measured by degranulation [16]. For decades, the presence of eosinophils (determined by total counts or percentages) has been the gold standard for measuring eosinophilic inflammation, a hallmark of allergic diseases such as asthma, AR, AD, and eosinophilic esophagitis. Atopic inflammation is a complex process involving several cytokines that affect eosinophil activation, proliferation, differentiation, and survival. Degranulation involves the extracellular release of 4 major proteins: EDN, ECP, major basic protein, and eosinophil peroxidase. All of these proteins are cytotoxic, anti-infectious, and, most importantly, involved in inflammation, airway remodeling, mucus hypersecretion, and lung epithelium damage [17]. ECP is the most studied and highly regarded biomarker for allergic diseases; however, in the past decade, EDN has garnered more attention owing to its superiority [18-20].

In direct comparison, EDN is a more accurate and easily utilized biomarker of eosinophilic inflammation in allergic diseases. A study of patients in the acute and stable phases of asthma evaluated serum EDN, serum ECP, and total eosinophil count as biomarkers. Only EDN levels were significantly elevated during the acute and stable phases of the disease (80 ng/mL and 42.9 ng/mL, respectively, compared to controls [20 ng/mL], P<0.0001), and only EDN was highly correlated with disease severity (r=0.850, P<0.0001) [20]. The EDN levels differed significantly between the acute and stable phases (P<0.001). There are many reasons for EDN’s superiority over EDN. EDN is more easily recovered from measuring instruments and cell surfaces than other eosinophil biomarkers because of its weaker electrical charge [21] (i.e., it is not "sticky"); therefore, storage tube type is not a concern. EDN recoverability was recently tested among 5 different polypropylene storage tubes, with a coefficient of variation of 1.5%–7.6%. This indicates low relative variability [22], which is critical for EDN's recoverability and reproducibility.

Other protein and peptide biomarkers are known to bind nonspecifically to different polypropylene storage tubes, which can distort data comparisons [23]. EDN is released from eosinophils with greater efficiency [24], increasing its recoverability. EDN can be easily obtained from many different bodily fluids, including blood, urine, sputum, nasal secretions, and bronchoalveolar lavage, and it is stable at ambient temperature or when refrigerated for at least 7 days. It is also stable when frozen at -20̊C or -80̊C for up to 1 year and even maintains this stability through multiple freeze/thaw cycles. EDN levels did not change in whole blood for 24 hours or do they change throughout the day. Unlike those of other eosinophilic inflammatory markers, EDN levels do not follow a circadian rhythm [22]. EDN has antiviral activity against respiratory infections that are strongly associated with allergic disease development later in life (e.g., RSV and hRV infections) [5,6]. EDN is released almost exclusively by eosinophils [22]; therefore, EDN levels directly reflect eosinophil activity. Peripheral blood eosinophils release 17%–28% of their total intracellular EDN, which is increased in asthmatic patients; in contrast, ECP is not [25], further adding to the superiority of EDN over ECP.

1. EDN in AD

Many genetic, environmental, and immunological predisposing factors are shared by allergic diseases, the key features of which are specific IgE generation, granulocyte activation, mucous production, and edema [17]. Eosinophils, the major effector cells in allergic diseases, are often elevated in number and activated. AD, the first step in atopic march, is usually diagnosed in the first 6 months of life prior to FA, asthma, and AR development [26,27]. AD is associated with peripheral eosinophilia and increased levels of eosinophil granule proteins such as EDN and ECP. Elevated serum EDN levels in young children with AD were also more significantly correlated with disease severity than elevated ECP levels [28].

Another study highlighted its measurability in several different specimen types and found that urinary EDN levels correlated well with AD severity and visual analog scale scores (itching, skin condition, skin symptoms, and total) [29]. An earlier study by the same research group [30] found that serum, plasma, and urine EDN concentrations were significantly lower when a patient's skin symptoms were mild versus severe (39.5 ng/mL vs. 62.8 ng/mL, P<0.01; 19.4 ng/mL vs. 28.0 ng/mL, P<0.01; 66.8 μg/mmol vs. 149.5 μg/mmol, P<0.01, respectively). A more recent study [18] reported increased EDN levels in schoolchildren with AD (110.8 ng/mL vs. 38.38 ng/mL in healthy controls). EDN levels were consistently elevated across the age range of the subjects (6–12 years), suggesting that EDN could be a useful screening biomarker throughout childhood [18].

2. EDN in FA

The development of FA is another step in the development of atopic march. Children with FA are at an increased risk of developing asthma, food-induced asthma episodes, and more severe asthma [31]. Elevated EDN levels have been observed in children with FA. In school-aged children (6–12 years of age), EDN levels were much higher in those with FA versus healthy controls (124.2 ng/mL vs. 38.38 ng/mL, respectively) [18]. Terashi et al. [32] recently investigated the association between blood EDN levels in preschool children with FA or AD and the onset of allergic airway diseases (bronchial asthma or AR). EDN levels were serially measured at 3 time points (<1, <2, and <3 years of age). At the first time point (<1 year of age), all study participants had similar EDN levels. However, at 2 and 3 years of age, children who eventually developed at least one allergic airway disease by 3 years of age had significantly higher EDN levels than those who did not develop any allergic airway disease (226.6 ng/mL vs. 65.0 ng/mL [P<0.01] at 2 years and 173.9 ng/mL vs. 62.7 ng/mL [P<0.01] at 3 years). This study's findings suggested that elevated EDN levels in young children with FA are associated with the development of allergic airway diseases later in life.

3. EDN in asthma

Asthma is the most common chronic disease in early childhood and the leading cause of morbidity. Lung function tests are an important part of an asthma diagnosis; however, they cannot be performed in children under 6 years of age. However, EDN levels can be measured at any age using serum, blood, or urine samples. The currently available EDN tests also require smaller-volume specimens, and children have a lower total blood volume and can be uncooperative with testing [33]. Several studies reported elevated EDN levels in children with asthma [18-20,25,34]. In a 2010 study by Kim et al. [20], EDN levels were measured in children during the acute and stable phases of asthma. EDN, ECP, and total eosinophil counts were higher in patients with acute asthma; however, in the stable phase, only EDN and ECP were elevated compared to those in controls. Patients with acute asthma were treated with albuterol and systemic corticosteroids, which reduced the median EDN level from 80 ng/mL (acute phase) to 42.9 ng/mL (stable phase).

Comparison of the acute and stable phases revealed that only EDN and total eosinophil counts differed significantly. When study subjects were subgrouped according to disease severity (mild, moderate, and severe), only EDN levels differed significantly among them. Using 46 ng/mL as the cutoff for an elevated EDN level compared controls, the positive predictive value (PPV) for asthma was 93%, negative predictive value (NPV) was 54%, sensitivity was 66%, and specificity was 89%. Other studies found that serum EDN levels were higher in atopic children with asthma than in nonatopic children with asthma and controls.

Serum EDN levels are also associated with bronchial hyperresponsiveness [34]. A more recent study found EDN levels were elevated in children with asthma and predictive of asthma (sensitivity, 81.3%; specificity, 87.1%; PPV, 90.7%; and NPV, 75.0%) using a cutoff value of 44.2 ng/mL compared to healthy controls [33]. In this study, EDN was measured using a commercially available enzymelinked immunosorbent assay kit, which determined a value of <2.5 hours. This highlights the utility of EDN measurements in the clinical setting; the results are obtained quickly so that treatment decisions can be made.

4. EDN in wheezing

EDN exhibits antiviral activity against common respiratory viruses such as hRV and RSV, which are strongly associated with asthma development and exacerbation [6]. There appears to be a subgroup of young children infected with one or more of these viruses who develop recurrent wheezing, and EDN measurements may aid the early identification of patients in this group. In a study of 200 infants hospitalized with RSV bronchiolitis, serum EDN levels at 3 months post–hospital admission correlated well with total wheezing episodes at 12 months post–hospital admission. Using 53 ng/mL (mean + 2 standard deviations) as the cutoff for an elevated EDN level, this biomarker demonstrated prognostic value for recurrent wheezing development: PPV, 57%; NPV, 76%; sensitivity, 72%; and specificity, 62% [35]. Elevated EDN levels may be a distinctive feature of respiratory wheezing. Children with respiratory infections and wheezing were compared to those with respiratory infections (e.g., pneumonia, common cold, and tonsillitis) but without wheezing. The serum EDN levels of the former were significantly higher than those of the latter (P<0.001) [36].

EDN levels were also used to monitor treatment in a study of RSV bronchiolitis [35]. After 3 months of treatment with montelukast, a leukotriene receptor antagonist, EDN levels decreased significantly from 59 to 43 ng/mL (P<0.01). The EDN levels also remained significantly lower than those in the placebo group despite treatment discontinuation (P<0.001).

5. EDN in AR

AR is often among the last allergic diseases to develop in atopic march and part of the United Airway Disease hypothesis, aka "one airway, one disease." [37] Anatomical, histological, epidemiologic, pathophysiologic, and clinical evidence supports the idea that asthma and AR can be manifestations of a single inflammatory process. The treatment of one condition can improve the symptoms of the other [37]. Few studies have been published on EDN levels in children with AR. An early study from the 1990s reported that EDN levels were elevated in children with seasonal AR [38], whereas a recent study found elevated EDN levels in children with AR versus those without allergic disease (91.3 ng/mL vs. 38.4 ng/mL, respectively) [18].

EDN levels reflect changes in disease severity. EDN levels significantly differed in a comparison of asymptomatic and symptomatic seasonal AR in the same patients (serum [28.0 ng/mL vs. 41.3 ng/mL, P<0.01], plasma [16.1 ng/mL vs. 21.8 ng/mL, P<0.05], and urine [46.6 μg/mmol vs. 65.1 μg/mmol P<0.01], respectively) [30].

EDN predicts allergic disease development

Table 1 summarizes published studies showing that increased EDN levels are associated with allergic disease development, specifically recurrent wheezing, asthma, and/or AR. Terashi et al. [32] measured EDN levels at 3 time points (<1, 2, and 3 years of age) in very young children with at least one FA. At 1 year of age, no difference was noted between the group that developed allergic diseases (bronchial asthma and/or AR) and the group that did not. However, at 2 and 3 years of age, the group that developed allergic disease had significantly elevated EDN levels (226.6 ng/mL vs. 65.0 ng/mL and 173.9 ng/mL vs. 62.7 ng/mL, respectively; P<0.01). The authors suggested that EDN is a useful biomarker for the development of allergic diseases.

Fardig et al. [39] measured EDN levels in very young children at 1 year (27.4 μg/L) and 3 years (20.1 μg/L) of age. The median EDN levels at 1 year were higher in children with wheezing (P=0.001), preschool asthma (P=0.025), allergic sensitization (P<0.001), and AD (P<0.002) than in nonatopic children (those with no history of asthma, wheezing, allergic sensitization, or AD between birth and 3 years of age). The median EDN levels at 3 years of age were higher in children with wheezing (P=0.016), preschool asthma (P<0.001), AD (P=0.001), and preschool asthma with allergic sensitization and/or AD (P<0.001). The allergic-sensitized children in this study had among the highest EDN levels at both 1 and 3 years of age, while those with preschool asthma and comorbidities of allergic sensitization and/or AD had among the highest EDN levels at 1 year of age, suggesting a phenotype in which eosinophil inflammation is established in infancy. The authors concluded that EDN, alone or in combination with other biomarkers, may be a useful tool in clinical practice.

A novel 2022 study by Sereme et al. [40] measured EDN in the stool of preterm neonates, who were followed up 1 year later for atopic disease development. During fetal life, the maternal microbiota produces compounds that are transferred to the fetus to generate innate immune cells [41]. Premature birth halts this process, leaving preterm infants vulnerable to diseases such as respiratory infections that negatively affect lung development [41-43]. Cohort studies reported that preterm children are at an increased risk of preschool wheezing and school-age asthma [44]. According to Sereme et al. [40], the presence of atopic conditions at 1 year of age was positively associated with EDN abundance from birth to 6 weeks of age. Specifically, EDN level was positively correlated (r=0.59) with the development of AD and cow's milk allergy within 1 year of age. The authors concluded that high EDN production during the first weeks of life may indicate an individual's risk of allergic disease development.

A study of infants hospitalized for respiratory diseases was conducted 1 year later. Children who went on to develop recurrent wheezing had much higher EDN levels on admission than those who did not (68.67 ng/mL vs. 27.36 ng/mL, respectively; P<0.001) [45]. The presence of eczema on admission was also associated with an increased risk of recurrent wheezing (OR, 4.313). Zhai et al. [45] concluded that serum EDN level had strong prognostic power for recurrent wheezing and was a predominant risk factor for recurrent wheezing in infants.

Chakraborty et al. [46] enrolled 156 young children (aged 6 months to 3 years) with wheezing in a longitudinal study. By 4 years of age, wheezing children who would develop asthma by 7 years of age had significantly elevated EDN levels compared to wheezing children who did not eventually develop asthma. EDN levels gradually increased from 3 years onward in the group that developed asthma by 7 years of age. The authors concluded that increased EDN levels in preschool-aged wheezers may be a biomarker of asthma development and could be used to enable an earlier clinical asthma diagnosis.

An earlier study by Kim et al. [35] found that elevated EDN levels after RSV bronchiolitis were significantly correlated with total wheezing episodes after 12 months of age. A total of 25.4% of children with EDN levels >53 ng/mL developed recurrent wheezing by 12 months of age. The authors concluded that EDN level may be an accurate biomarker for diagnosing, treating, and monitoring eosinophil-associated diseases in young children. Furthermore, EDN has predictive value for the development of recurrent wheezing after RSV bronchiolitis. This information would be especially helpful since early diagnosis and treatment may slow allergic march progression.

Conclusions

Allergic diseases are common and on the rise; therefore, it is important to identify objective common measures of the underlying pathophysiology. EDN, an eosinophilic granule protein, has demonstrated reliability and accuracy as a biomarker of eosinophilic inflammation. EDN levels can be elevated from a very young age, even before an allergic disease is diagnosed. Moreover, they are often elevated in diseases that comprise atopic march and can even predict the development of allergic diseases later in life. The EDN can be easily obtained from a number of different specimen types and measured at any age. Therefore, its measurement should be considered a welcome addition to the clinician's arsenal for the prognosis, diagnosis, treatment, and monitoring of the allergic conditions comprising atopic march.

Footnotes

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author Contribution

ZC contributed to the conception, literature review, initial draft, editing, final draft, and submission of the manuscript. CKK contributed to the conception, literature review, initial draft, editing, final draft, and submission of the manuscript.

Fig. 1.

Main steps of allergic march and age at onset.

Table 1.

Studies of elevated eosinophil-derived neurotoxin levels and allergic disease development

Study	Subjects	Specimen type	Disease predicted	Outcome
Terashi [32] (2023)	123	Serum	AR, asthma	High EDN levels in patients with FA/AD associated with asthma and/or AR onset later in life
Fardig [39] (2023)	1,233	Serum	Asthma	Higher EDN levels at 1 and 3 yr associated with preschool asthma
Sereme [40] (2022)	43	Feces	FA, AD	Elevated neonatal EDN associated with atopic conditions at 1 yr of age
Zhai [45] (2019)	145	Serum	Recurrent wheezing	Elevated EDN levels at 3 yr associated with recurrent wheezing development 1 yr later; eczema at 3 yr associated with recurrent wheezing development 1 yr later
Chakraborty [46] (2022)	258	Serum	Asthma	At 4 yr, wheezing children that developed asthma by age 7 had higher EDN levels than wheezing children that did not develop asthma; from 3-yr to 7-yr EDN levels gradually increased in children that developed asthma by age 7
Kim [35] (2013)	178	Serum	Recurrent wheezing	EDN levels at 3 mo after bronchiolitis correlated with total number of wheezing episodes at 12 mo; 73% of children with EDN > 100 ng/mL at 3 mo developed recurrent wheezing

AR, allergic rhinitis; EDN, eosinophil-derived neurotoxin; FA, food allergy; AD, atopic dermatitis.

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