Respiratory Medicine 123 (2017) 71e78

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Respiratory Medicine
journal homepage: www.elsevier.com/locate/rmed

Association of interleukin-25 levels with development of aspirin

induced respiratory diseases
Jong-Uk Lee a, 1, Hun Soo Chang a, b, 1, Hyeon Ju Lee a, Da-Jeong Bae a, Ji-Hye Son a,
Jong-Sook Park b, Jae Sung Choi c, Hun Gyu Hwang d, Choon-Sik Park a, b, *
a
Department of Interdisciplinary Program in Biomedical Science Major, Soonchunhyang Graduate School, Bucheon, Republic of Korea
b
Genome Research Center, Division of Allergy and Respiratory Medicine, Soonchunhyang University, Bucheon Hospital, Republic of Korea
c
Division of Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University, Cheonan Hospital, Republic of Korea
d
Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University, Gumi Hospital, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Background: Aspirin-exacerbated respiratory diseases (AERD) are caused by ingestion of non-steroidal
Received 11 September 2016 anti-inflammatory drugs and are characterized by acute bronchospasms and marked infiltration of eo-
Received in revised form sinophils, the latter being attributable to altered synthesis of cysteinyl leukotrienes (LT) and prosta-
21 November 2016
glandins (PG). Recently, the innate Th2 response is revealed to induce eosinophil infiltration in allergic
Accepted 22 November 2016
inflammation, however the role of the innate Th2 response has not been studies in AERD. Thus, we
Available online 24 November 2016
evaluated the relationship between the innate Th2 cytokines including IL-25, thymic stromal lympho-
poietin (TSLP) and IL-33 and the development of AERD.
Keywords:
Asthma
Methods and materials: Plasma IL-25, IL-33, and TSLP levels were measured before and after aspirin
Aspirin challenge in subjects with AERD (n ¼ 25) and aspirin-tolerant asthma (ATA, n ¼ 25) by enzyme-linked
IL-25 immunosorbent assay (ELISA). Pre and post-aspirin challenge levels of LTC4 and PGD2 were measured
Innate immune response using ELISA.
Results: Basal plasma IL-25 levels were significantly higher in AERD group than in normal controls and in
ATA group (p ¼ 0.025 and 0.031, respectively). IL-33 and TSLP levels were comparable in the AERD and
ATA groups. After the aspirin challenge, the IL-25 levels were markedly decreased in the ATA group
(p ¼ 0.024), while not changed in the AERD group. The post-challenge IL-25 levels of all asthmatic
subjects were significantly correlated with aspirin challenge - induced declines in FEV1 (r ¼ 0.357,
p ¼ 0.011), but not with basal and post challenge LTC4 and PGD2 levels.
Conclusions: IL-25 is associated with bronchospasm after aspirin challenge, possibly via mechanisms
other than altered LTC4 and PGD2 production.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction respiratory reactions on ingestion of aspirin and other NSAIDs that

Aspirin-exacerbated respiratory disease (AERD) refers to the
development of non-allergic hypersensitivity reactions by asth-
matics following the ingestion of aspirin or other non-steroidal
anti-inflammatory drugs (NSAIDs) [1]. The syndrome is character-
ized by clinical manifestations of nasal polyps, chronic hypertro-
phic eosinophilic sinusitis, asthma, and noneIgE-mediated
* Corresponding author. Division of Allergy and Respiratory Medicine, Dept. of
Internal Medicine, Soonchunhyang Univ., Bucheon Hospital, 1174, Jung-Dong,
Wonmi-Ku, Bucheon, Kyeonggi-Do, 420-767, Republic of Korea.
E-mail address: mdcspark@daum.net (C.-S. Park).
1
Jong-Uk Lee and Hun Soo Chang contributed equally as the first authors.
inhibit cyclooxygenase-1 (COX-1) [2]. AERD is associated with
eosinophilic airway inflammation and ongoing activation of mast
cells with systemic release of multiple mediators, including leu-
kotrienes, prostaglandins, and tryptase [1]. One of the main un-
derlying mechanisms involves overproduction of cysteinyl
leukotrienes (cysLTs) and overexpression of the cysLT receptor by
inflammatory cells of the respiratory tract [3]. In addition to the
increased levels of cysLTs in various biological fluids, including
saliva, induced sputum, blood, and urine [4], dysregulation of
arachidonic acid (AA) metabolism reduces prostaglandin E2 (PGE2)
synthesis and increases prostaglandin D2 (PGD2) production in
patients with AERD compared with those in patients with aspirin-
tolerant asthma (ATA) [5].

http://dx.doi.org/10.1016/j.rmed.2016.11.020
0954-6111/© 2016 Elsevier Ltd. All rights reserved.
72 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78
Histologically, AERD is characterized by extensive eosinophilic
inflammation in the sino-nasal and bronchial mucosae, along with
the presence of degranulated mast cells [6]. Infiltration of eosino-
phils and mast cells is accompanied by elevated levels of the
eosinophil-active cytokines IL-5 and eotaxin and activated T-helper
type 2 (Th2) cytokines in the airways of AERD patients [7e9]. As in
asthma, a shift from a T-helper type 1 (Th1) to a Th2 immune
response is the major mechanism of AERD. This results in the
overproduction of various cytokinesdsuch as interleukin 4 (IL-4),
IL-5, and IL-13dand underproduction of the Th1-type cytokine
interferon-gamma (IFN-g) [10]. Recently, innate Th2 immune
response has been well documented in asthma and allergic
inflammation [11,12]. However, the role of the innate Th2 has not
been studied in AERD up to data.
Epithelial cell-derived cytokines, including thymic stromal
lymphopoietin (TSLP), IL-25, and IL-33 influence innate immunity
in several inflammatory diseases, including inflammatory bowel
diseases, asthma, and atopic dermatitis [13]. TSLP can activate mast
cells to generate type 2 cytokines [14], which stimulate eosinophils
[15], basophils [16], type 2 innate lymphoid cells (ILC2s) [17], and
CD34-positive hematopoietic progenitor cells [18] in conjunction
with IL-1, TNFa, or IL-33 [14,19]. IL-33 alone is capable of acting on
mast cells to induce expression of Th2 cytokines as well as of
increasing the survival and sensitization of mast cells to TSLP [19].
IL-25 also induces expansion of a non-B, non-T ckit þ cell popula-
tion in vivo. Additionally, this presumed mast cell progenitor pop-
ulation expresses Th2 cytokines in response to stimulation by IL-25
[20].
Nasal polyps are outgrowths of inflamed sino-nasal mucosa that
occur in patients with chronic rhinosinusitis. They are densely
infiltrated by eosinophils, activated mast cells, and large numbers
of ILC2s [21,22]. Recently, the TSLP level was reported to be elevated
in nasal polyps of AERD patients and to stimulate mast cells to
produce a large quantity of PGD2 [23]. PGD2 is the preferred ligand
for the chemoattractant receptor homologue expressed by TH2
cells (CRTH2), which is expressed by Th2 cells [24] eosinophils,
basophils [24,25], and ILC2s [26]. The IL-33 level is also increased in
the nasal polyps of AERD patients, which is driven by cysLTs [27].
Accordingly, IL-33, IL-25, and TSLP may stimulate ILC2 to produce
Th2 cytokines in conjunction with PGD2 and cysLTs in AERD.
However, the contribution of each cytokine to the development of
AERD has not been evaluated to date. In the present study, plasma
IL-33, IL-25, and TSLP levels were compared before and after aspirin
challenge in subjects with AERD and ATA, and were analyzed in
terms of their association with PGD2 and LTE4 levels.
2. Materials and methods
2.1. Study subjects
All patients were diagnosed by physicians and met the criteria
for asthma of the Global Initiative for Asthma (GINA) guidelines. All
patients had a history of dyspnea and wheezing during the previ-
ous 12 months plus one of the following: (1) >15% increase in FEV1
or >12% increase plus 200 mL following inhalation of a short-acting
bronchodilator, (2) <10 mg/mL PC20 methacholine, and (3) >20%
increase in FEV1 following 2 weeks of treatment with inhaled
steroids and long-acting bronchodilators. Current smokers and ex-
smokers with more than 10 pack year were excluded. At the
baseline visit, demographic information, such as enrollment age,
sex, BMI, onset age of asthma, asthma duration, smoking amount,
was collected. All patients underwent a standardized assessment,
which included analyses of the induced sputum, complete blood
cell count with differential counts, total IgE, chest radiography,
spirometry, and allergy skin prick tests with 24 common inhalant
allergens (Bencard Co., Brentford, UK). Atopy was defined as a
wheal reaction equal to or greater than that to histamine or at least
3 mm in diameter over that of saline control. Total IgE was
measured using the CAP system (Pharmacia Diagnostics, Uppsala,
Sweden). The asthma patients had not experienced exacerbations
of asthma or respiratory tract infections during the 6 weeks pre-
ceding the oral aspirin challenge (OAC). The OAC was carried out
using increasing doses of aspirin [28,29]. Briefly, patients with a
history of aspirin hypersensitivity were administered 30 mg orally.
Respiratory and nasal symptoms, blood pressure, external signs
(urticaria and angioedema), and FEV1 were documented at 30-min
intervals for a period of 1.5 h. In the absence of any symptom or sign
suggesting an adverse reaction after 1.5 h, increasing doses of
aspirin (60, 100, 300, and 400 mg) were administered until the
patient developed a reaction, and the measurements were repeated
at 1 h intervals. Those having no history were started on 100 mg of
aspirin, which was gradually increased to 200, 350, and 450 mg
until the patient developed a reaction. If no reaction had occurred
4 h after the final dose, the test result was deemed to be negative.
Aspirin-induced bronchospasm, reflected by a decline (%) in FEV1,
was calculated as the pre-challenge FEV1 minus the post-challenge
FEV1 divided by the pre-challenge FEV1. OAC reactions were cate-
gorized into the following two groups: (1) 15% or greater decrease
in FEV1 or nasal reactions, such as rhinorrhea and nasal congestion
(AERD); and (2) less than a 15% decrease in FEV1 without naso-
ocular or cutaneous reactions (ATA). Peripheral venous blood was
collected before and after the aspirin challenge. None of the study
subjects had been treated with the cysteinyl leukotriene (cysLT) 1
receptor blocker montelukastor or the 5-lipoxygenase (5-LO) in-
hibitor zileuton before the challenge. The patients' spouses and
general population were recruited as normal controls (NCs). The
NCs had no respiratory symptoms, as determined by a screening
questionnaire [30], had a predicted FEV1 and FVC >80%, and had
normal chest radiogram results. All study subjects were Korean and
provided informed written consent to participate in the study.
Plasma from subjects with AERD (n ¼ 25), ATA (n ¼ 25), and normal
controls (NCs) (n ¼ 20) was obtained from a biobank at Soon-
chunhyang University Hospital, Bucheon, Korea, after approval of
the protocol by the Ethics Committee of Soonchunhyang University
Hospital (approval no. SCHBC 2015-06-018-001, schbc-biobank-
2015-011-01).
2.2. Measurement of plasma IL-25, IL-33, TSLP, LTC4 and PGD2
levels
IL-25, IL-33, and TSLP levels were measured using quantitative
sandwich enzyme immunoassay kits: IL-25 (Busterbio, CA, USA)
and IL-33 and TSLP (R&D Systems, Minneapolis, MN) according to
the manufacturer's recommendations. The lower limits of detection
were 10 pg/mL (IL-25), 0.52 pg/mL (IL-33), and 3.46 pg/mL (TSLP).
Results below these thresholds were assigned a value of 0 pg/mL.
Plasma LTC4 and PGD2 levels were measured using an enzyme
immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) accord-
ing to the manufacturer's recommendations. The inter- and intra-
assay coefficients of variability for all assays were less than 15%.
2.3. Statistical analysis
Data analysis was performed using the statistical software
package SPSS ver. 20.0. The normality of the distribution of data
was evaluated by means of a ShapiroeWilk test. Normally distrib-
uted data are presented as means (standard errors), and skewed
data are presented as medians (interquartile ranges). Comparisons
were performed using the KruskaleWallis test and a post hoc
analysis; a ManneWhitney U-test was conducted to evaluate
 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78 73

differences in nonparametric variables. One-way ANOVA and a post
hoc Tukey's range test were applied to normally distributed data.
The Wilcoxon signed-rank test was applied to the paired samples
before and after the aspirin challenge. Nominal variables were
analyzed by Pearson's chi-squared test. Correlations of IL-25, IL-33,
and TSLP levels with other parameters were evaluated by Spear-
man's correlation coefficient analysis. A value of p < 0.05 was
considered as statistical significance.
3. Results
3.1. Characteristics of patients participating in the study
The clinical and laboratory profiles of the study subjects are
presented in Table 1. Subjects with AERD and those with ATA had
lower values of FEV1 and FVC and elevated levels of IgE and blood
eosinophils compared with NCs. There were no significant differ-
ences in the other clinical parameters except PC20 methacholine
between the AERD and ATA patients.
3.4. Baseline and post-aspirin challenge plasma LTC4 and PGD2
levels
LTC4 and PGD2 were detected in the plasma of all study sub-
jects. The plasma LTC4 level was significantly higher in subjects
with AERD [1258.34 (319.65e1550.74) pg/mL] than in NCs [73.43
(10.0e250.85) pg/mL, p ¼ 4.81  105] and in those with ATA
[489.28 (100.85e984.44) pg/mL, p ¼ 0.042]. The plasma PGD2 level
was significantly higher in subjects with AERD [179.03
(46.66e303.97) pg/mL] and those with ATA [199.36 (49.12e407.19)
pg/mL] than in NCs [69.04 (26.76e158.85) pg/mL, p ¼ 0.04 and
0.009, respectively]. The LTC4/PGD2 ratio was significantly higher
in subjects with AERD [6.36 (1.02e26.71) pg/mL] and those with
ATA [3.67 (0.39e7.62) pg/mL] than in NCs [1.08 (0.19e2.33) pg/mL]
(p ¼ 0.001 and p ¼ 0.038, respectively) (Fig. 3).
Aspirin challenge significantly increased LTC4 and PGD2 levels
both in AERD and in ATA groups (Fig. 4). Post-challenge LTC4 levels
were significantly increased in AERD compared to those in ATA
(p ¼ 0.029), while post-challenge PGD2 levels were not different
between the two groups (p > 0.05).

3.2. Baseline plasma IL-25, IL-33, and TSLP levels
IL-25 was detected in the plasma of all of NCs, in 23 of 25 the
ATA subjects, and in 23 of 25 the AERD subjects. The IL-25 level was
significantly higher in subjects with AERD [101.13 (54.23e171.83)
pg/mL] than in NCs [64.19 (47.09e81.45) pg/mL, p ¼ 0.025] and
subjects with ATA [41.38 (17.94e109.54) pg/mL, p ¼ 0.031]. IL-33
was detected in six subjects with ATA and in four with AERD.
TSLP was detected in two subjects with ATA and in three with
AERD. There was no difference in the IL-33 and TSLP levels of the
AERD and ATA groups (Fig. 1).
3.3. Changes in IL25, IL-33, and TSLP levels after aspirin challenge
The IL-25 levels of subjects with ATA were significantly
decreased after the aspirin challenge compared with those at
baseline [41.38 (17.94e109.54) vs. 34.92 (0.0e81.18) pg/mL,
p ¼ 0.024], But was no decreased in subjects with AERD [101.13
(54.23e171.83) vs. 85.86 (55.49e219.21) pg/mL], but statistically
insignificant (p ¼ 0.267). The IL-33 and TSLP levels after the aspirin
challenge were unchanged compared with the baseline values
(Fig. 2).
3.5. Correlations of IL-25 level with aspirin-induced declines in
FEV1, and other parameters including LTC4, and PGD2 in asthmatics
In all asthmatics, the post-aspirin challenge IL-25 levels were
analyzed for correlation with LTC4 (pg/mL), PGD2 (pg/mL) LTC4/
PGD2 ratio, IgE (IU/mL), Blood eosinophils (%), FVC(%, pred),
FEV1(%, pred) and % decline of FEV1 by aspirin provoction. Among
them, the post-aspirin challenge IL-25 levels and LTC4 levels were
significantly correlated with the aspirin-induced decline in FEV1
(Fig. 5A and B) (p ¼ 0.011, r ¼ 0.357 and p ¼ 0.021, r ¼ 0.327, n ¼ 50),
while the pre- and post-challenge IL-25, LTC4, PGD2 levels and
LTC4/PGD2 ratio were not correlated with the other parameters
(Table 2 and 3). In the subgroup analysis, there was no significant
correaltion among the parameters in ATA or AERD groups
(Supplementary Table 1 and 2).
4. Discussion
In the present study, We demonstrated that the plasma IL-25
level significantly increases in AERD compared to normal controls
and ATA group. Moreover, the magnitude of the elevation was
greater in the AERD than in the ATA group. Interestingly, IL-25
levels were markedly attenuated by the aspirin challenge in the

Table 1
Clinical profile of study subjects.

Description NC Asthma p-value Asthma

AERD ATA p-value

Number of subjects 39 50 25 25
Age, median (years) 58.5(29.0e63.0) 54.5(38.3e65.0) 0.319 56.0(40.0e65.5) 54.0(36.0e64.5) 0.931
Onset age, median (years) ND 50.5(29.3e58.8) ND 48.5(27.3e57.5) 56.0(35.5e60.0) 0.385
Duration, median (years) ND 8.0(6.25e16.0) ND 6.5(6.0e11.5) 10.0(6.25e16) 0.714
SEX (Male/Female) 10/10 15/35 0.115 5/20 10/15 0.123
Non smoker/ex smoker(%) 94.88/5.12 92.0/8.0 0.787 92.0/8.0 92.0/8.0 0.637
BMI(kg/m2) 23.41 ± 3.36 24.18 ± 4.16 0.429 23.99 ± 3.93 24.36 ± 4.46 0.760
% decline of FEV1 by aspirin provocation ND 18.10 ± 26.46 ND 42.08 ± 14.92 5.88 ± 2.98 1.2  109
Blood Eosinophill (%) 0.38 ± 4.43 2.75 ± 4.75y 0.018 1.88 ± 4.42y 3.63 ± 6.18 0.540
FVC %, predicted 90.75 ± 12.18 84.02 ± 10.97 0.039 83.8 ± 11.32 84.24 ± 10.84 0.961
FEV1%, predicted 101.35 ± 10.11 83.44 ± 16.18 3.9  105 82.84 ± 16.49 84.04 ± 16.17 0.907
PC20,methacholine (mg/mL) ND 7.75 ± 9.72 ND 5.16 ± 8.48 10.14 ± 10.32 0.047
Total IgE (IU/mL) 38.82 ± 22.32 487.46 ± 783.41 0.8  104 443.35 ± 472.79 531.58 ± 1012.64 0.273

Definitions of abbreviations: NCs, normal controls; AERD, aspirin-exacerbated respiratory disease; ASA, aspirin; ATA, aspirin-tolerant asthma.
Differences in patient characteristics [shown as medians (interquartile ranges)] among the NC, AERD, and ATA groups were evaluated using the KruskaleWallis test and a post
hoc analysis; a ManneWhitney U-test. Differences in patient characteristics [shown as means ± standard error of the mean (SEM)] among the groups were evaluated by one-
way ANOVA and Tukey's honestly significant difference post hoc test. Significance: compared with NCs, yp < 0.05; compared with AERD subjects, *p < 0.05.
74 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78

Fig. 1. Baseline plasma IL-25, IL-33, and TSLP levels. (A) IL-25 protein was detected in all 20 NCs, in 23 of 25 ATA subjects, and in 23 of 25 AERD subjects. Open and closed circles
indicate IL-25 protein levels and those below the lower limit of detection (>10 pg/mL), respectively. (B) IL-33 protein was detected in six subjects with ATA and in three with AERD.
The open and closed circles indicate IL-33 protein levels and those below the lower limit of detection (>0.519 pg/mL), respectively. (C) TSLP was detected in two subjects with ATA
and in three with AERD. Open and closed circles indicate TSLP protein levels and those below the lower limit of detection (>3.46 pg/mL), respectively. Comparisons were performed
using the KruskaleWallis test and a post hoc analysis; a ManneWhitney U-test was conducted to evaluate nonparametric variables. Data are presented as box plots of median values
and interquartile ranges. A value of p < 0.05 was considered to indicate statistical significance.

Fig. 2. Changes in IL-25, IL-33, and TSLP levels in plasma after aspirin challenge in 25 patients with AERD and in 25 patients with ATA. (A) IL-25, (B) IL-33, and (C) TSLP levels.
Comparisons were performed using the Wilcoxon signed-rank test. A value of p < 0.05 was considered to indicate statistical significance.

Fig. 3. Baseline plasma LTC4, PGD2, and LTC4/PGD2 levels. (A) LTC4 and (B) PGD2 were detected in the plasma of all NCs, ATA subjects, and AERD subjects. Detection limits of LTC4
and PGD2 levels were 10 pg/mL and 12.35 pg/mL, respectively. (C) LTC/PGD4 ratios of NCs and subjects with ATA and AERD. Comparisons were performed using the KruskaleWallis
test and a post hoc analysis; a ManneWhitney U-test was conducted to evaluate differences in nonparametric variables. Data are presented as box plots of median values and
interquartile ranges. A value of p < 0.05 was considered to indicate statistical significance.

ATA but not significantly in the AERD group. These data indicate an stable in a certain percentage of AERD even after aspirin challenge.
enhanced innate Th2 response in AERD patients, which may be Additionally, the IL-25 levels after the aspirin challenge were
 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78 75

Fig. 4. Changes in LTC4 and PGD2 levels in plasma after aspirin challenge in 25 patients with AERD and in 25 with ATA. (A) LTC4 (B) PGD2 levels. Comparisons between before and
after aspirin challenge in each group were performed using the Wilcoxon signed-rank test. Comparisons between ATA and AERD were performed using the Mann-Whitney U test. A
value of p < 0.05 was considered to indicate statistical significance.

strongly correlated with the aspirin-induced decline in FEV1, sug-
gesting that IL-25 may be associated with the aspirin - induced
bronchospasm in asthmatics. Systemic release of multiple media-
tors, including leukotrienes, prostaglandins, and tryptase, due to
activation of mast cells is a characteristic finding of AERD [5].
Recently, epithelial cell-derived cytokines were reported to influ-
ence innate Th2 immune responses in asthma patients [13]. TSLP
and IL-33 activate mast cells and type 2 innate lymphoid cells to
produce type 2 cytokines [14] (ILC2s) [14,17,19]. Levels of the active
form of TSLP protein increases in nasal polyps from patients with
AERD compared with those in aspirin-tolerant control subjects
[23]. IL-33 is strongly expressed in both refractory nasal polyposis
[31] and severe asthma [32] and robustly expressed in the epithe-
lium of nasal polyps of AERD patients [27]. In our study, however,
TSLP and IL-33 were detected in the plasma of only a small pro-
portion of AERD and ATA subjects. These data suggest that the
elevated expression of TSLP and IL-33 in the nasal polyps of AERD
patients might not reflect their levels in peripheral blood.
In the present study, the plasma LTC4 level was significantly
higher in AERD compared with in ATA subjects; in contrast, the
plasma PGD2 levels of the two groups were comparable. AERD is
characterized by dysregulation of arachidonic acid (AA) meta-
bolism: increased levels of cysLTs [4] and reduced prostaglandin E2
(PGE2) synthesis [5]. In case of PGD2 production in AERD, levels of
urinary PGD2 metabolites (9a and 11b-PGF2) are increased after the
aspirin challenge in AERD [33]. TSLP directly affects the production
of PGD2. Nasal polyp TSLP mRNA expression is strongly correlated
with that of the mRNA encoding hematopoietic PGD2 synthase
[23]. Additionally, recombinant TSLP induces PGD2 production by
cultured human mast cells [23]. CysLTs drive IL-33 overproduction
[27], which stimulates ILC2 cells to produce Th2 cytokines in
conjunction with PGD2 and cysLTs. However, the increased
expression of these two cytokines did not reflect their levels in the
peripheral blood of ARED subjects in our study. We did not measure
PGD2 levels in urine because urine samples were not available,
which appears a limitation of our study.
In vitro-differentiated Th2 cells and mast cells produce IL-25
[34] [35]. IL-25 also induces the expansion of mast cell progenitor
population [20], which generate cysLTs and PGD2 [36]. Over pro-
duction of cysLTs and PGD2 after aspirin challenge in AERD may
attribute to the enhanced IL-25 generation. However, the lack of a
correlation between the plasma levels of IL-25 with those of LTC4

Fig. 5. Correlation of post challenge IL-25 and LTC4 protein levels in total asthma subjects with % decline in FEV1 due to aspirin provocation (p ¼ 0.011, r ¼ 0.357 and p ¼ 0.021,
r ¼ 0.327, n ¼ 50). Correlation was assessed using Spearman's correlation coefficient analysis.
76 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78

Table 2
Correlations of IL-25 levels in pre-and post-aspirin challenge test with other laboratory parameters in asthmatics.

Parameter N IL-25 (pre-challenge) IL-25 (post-challenge)

Correlation coefficient p-value Correlation coefficient p-value

LTC4 (pg/mL) (pre-) 50 0.033 0.821 0.047 0.744

LTC4 (pg/mL) (post-) 50 0.021 0.884 0.114 0.431
PGD2 (pg/mL) (pre-) 50 0.088 0.541 0.140 0.333
PGD2 (pg/mL) (post-) 50 0.121 0.401 0.139 0.336
LTC4/PGD2 ratio (pre-) 50 0.076 0.601 0.091 0.529
LTC4/PGD2 ratio (post-) 50 0.099 0.495 0,165 0.253
IgE (IU/mL) 50 0.056 0.702 0.004 0.978
Blood eosinophils (%) 50 0.069 0.632 0.071 0.624
FVC (%, pred) 50 0.159 0.271 0.230 0.109
FEV1 (%, pred) 50 0.286 0.540 0.235 0.100
% decline of FEV1 by aspirin provocation 50 0.211 0.140 0.357 0.011

Correlations between IL-25 levels and clinical outcomes were evaluated using Spearman's correlation coefficient analysis. Values of p < 0.05 were considered to indicate
statistical significance.

Table 3
Correlations of LTC4, PGD2, and LTC4/PGD2 levels in pre- and post-aspirin challenge with other laboratory parameters in asthmatics.

Parameter N LTC4 (pg/mL) Prostaglandin D2 (pg/mL) LTC4/PGD2 (pg/mL)

(pre-challenge) (post-challenge) (pre-challenge) (post-challenge) (pre-challenge) (post-challenge)

Correlation p- Correlation p- Correlation p- Correlation p- Correlation p- Correlation p-

coefficient value coefficient value coefficient value coefficient value coefficient value coefficient value

IgE (IU/mL) 50 0.002 0.991 0.002 0.989 0.098 0.498 0.122 0.398 0.082 0.571 0.093 0.519
Blood eosinophils (%) 50 0.186 0.195 0.191 0.185 0.138 0.338 0.129 0.372 0.020 0.889 0.115 0.425
FVC (%, pred) 50 0.121 0.404 0.228 0.411 0.002 0.991 0.244 0.088 0.128 0.377 0.277 0.051
FEV1 (%, pred) 50 0.028 0.846 0.130 0.367 0.047 0.745 0.273 0.055 0.061 0.674 0.231 0.106
% decline of FEV1 by aspirin 50 0.156 0.280 0.327 0.021 0.114 0.432 0.077 0.596 0.208 0.148 0.252 0.078
provocation

Correlations between LTC4, PGD2, and LTC4/PGD2 levels and clinical outcomes were evaluated using Spearman's correlation coefficient analysis. Values of p < 0.05 were
considered to indicate statistical significance.

and PGD2 in the present study may suggest the absence of rela- of IL-25 levels after aspirin challenge may be attributed to the
tionship between IL-25 and LTC4 or PGD2 in the peripheral blood. difference of these genetic variant.
Interestingly, IL-25 levels were attenuated by the aspirin challenge The present study was limited by the small number of AERD and
in ATA subjects but not in those with AERD. Aspirin regulate a ATA subjects. However, changes in the IL-25 levels between before
complex network of biochemical and cellular events. We reported and after the aspirin challenge were detected in all of 50 asth-
previously that aspirin upregulates IL-4 production to a twofold matics, suggesting that the change was meaningful. A second lim-
greater level in the peripheral blood mononuclear cells from pa- itation was that we did not measure cytokine levels in the airway,
tients with AERD compared with in those from patients with ATA such as in nasal lavage fluid or sputum. Additionally, we did not
[37]. IL-4 transcription is increased in the nasal mucosa of AERD determine whether plasma IL-25 was originated from the nasal and
patients [38]. IL-4 is responsible for some of the characteristic bronchial mucosa or other sites due to lack of suitable samples from
eicosanoid dysregulation reported in AERD, such as overexpression the same subjects.
of 15 lipoxygenase (15-LOX), prostaglandin D synthase (PGDS),
leukotriene C4 synthase (LTC4S), leukotriene A4 hydrolase (LTA4H),
5. Conclusion
and the cysLT receptors. Inhibition of cyclooxygenase 2 (COX-2),
prostaglandin E synthase (PGES), and its receptor prostanoid E re-
In the present study, plasma IL-33, IL-25, and TSLP levels were
ceptor 2 (EP2) is also attributed to IL-4 [39]. Aspirin, particularly at
compared before and after the aspirin challenge in subjects with
high doses, has both inhibitory and stimulatory effects on notable
AERD and those with ATA to test the hypothesis that the innate Th2
signaling molecules. These include NF-kB [40], ERK1/2 [41], STAT6
response is related to the development of AERD. The baseline
[42,43], p38 [44], JNK [45] and NFAT1, and NFAT2 [46]. Notably,
plasma IL-25 levels were significantly higher in subjects with AERD
aspirin itself has been shown to increase intracellular Ca2þ levels,
than in NCs and subjects with ATA. Furthermore, the IL-25 levels
transiently upregulate kinase C protein levels in T cells [47,48], and
were markedly decreased in ATA subjects compared with those
activate p38 and JNK [49,50]. We performed an in silico analysis of
before the aspirin challenge, whereas the levels were minimally
transcription factors on the promoter region of IL-25 and found
decreased in AERD subjects. The IL-25 levels were significantly
predicted sites for c-Jun, Elk-1, NFAT1, and NFAT2 (Supplementary
correlated with the decline in FEV1 after the aspirin challenge in all
Table 3). Thus, we analyzed associations of IL-25 levels with ge-
asthmatic subjects. These data suggest that IL-25 in peripheral
netic variants on these transcription factors using the previous
bloods may be associated with the development of AERD and the
GWAS data [51] [52] and an exome chip data [53] in 22 subjects.
aspirin challenge induced - airway obstruction.
Interestingly, rs4912202 and rs12405774 on c-Jun/AP-1, rs183374,
rs383068, rs546316 on NFAT2 showed significant association of
plasma IL-25 levels with p-values less than 107in a recessive mode Author disclosure
(Supplementary Table 4) This data indicate that different response
None of the authors has a financial relationship with a
 J.-U. Lee et al. / Respiratory Medicine 123 (2017) 71e78
commercial entity that has an interest in the subject of this
manuscript.
Author contributions
JU Lee, HS Chang and CS Park conceived and designed the ex-
periments, and wrote the manuscript; HJ Lee, DJ Bae and JH Son
performed experiments; JS Park, JS Choi, HG Whang performed oral
aspirin challenge and collected blood samples. All authors revised
and approved the final version of the manuscript.
Conflict of interest
There are no financial or other issues that might lead to conflict
of interest.
Acknowledgments
This study was supported by a grant from the National Research
Foundation of Korea funded by the Korean Government
(2013R1A2A2A01068077) and a research grant from Soon-
chunhyang Univ. to CS Park. Plasma samples were generously
provided by the Soonchunhyang University Bucheon Hospital Bio-
bank, a member of the National Biobank of Korea, supported by the
Ministry of Health, Welfare and Family Affairs, Republic of Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.rmed.2016.11.020.
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