1
2
3
4
YKL-40 is correlated to FEV1 and asthma control test (ACT) in asthmatic
patients: influence of treatment
5
Abstract
6
Background: YKL-40, also called chitinase-3-like-1 (CHI3L1) protein, has recently
7
shown its potential as a marker for asthma. The aims of present study were to
8
investigate whether the serum YKL-40 levels are stable or decreased in patients with
9
asthma after appropriate treatment, and to evaluate the correlation of YKL-40 levels
10
to lung function and asthma control test (ACT).
11
Methods: 103 asthmatic patients (mean age 33.1 ± 0.9 years) with newly diagnosed
12
asthma were enrolled in our study. Patients underwent a detailed clinical examination
13
and completed the ACT questionnaire, serum YKL-40 measurement, and spirometry
14
before (visit 1) and 8 weeks after initiation of treatment (visit 2).
15
Results: At visit 2, the median serum YKL-40 level was significantly decreased
16
compared with those at visit 1 (75.2 [55.8-86.8] ng/ml versus 54.5 [46.4-58.4] ng/ml,
17
p<0.001). Serum YKL-40 level was found to have a negative correlation with %FEV1
18
(r=-0.37, p<0.001) and ACT score (r=-0.26, p=0.007) at visit 1. The change in serum
19
YKL-40 levels between two visits were significantly correlated to changes in FEV1
20
(r=-0.28, p=0.006) and ACT score (r=-0.22, p=0.037). Patients with elevated levels of
21
YKL-40 had significantly greater corticosteroid use than patients with lower levels.
22
Conclusions: YKL-40 was found in decreased quantities in the serum of asthmatic
23
patients after appropriate treatment, and its levels correlated with improvements
24
in %FEV1 and ACT. High levels of serum YKL-40 may be refractory to current
25
asthma treatments.
26
27
Trial registration: ChiCTR-OCC-13003316
28
Keywords: asthma, CHI3L1, exacerbation, YKL-40
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Introduction
35
36
Asthma is a common chronic disease characterized by acute inflammation of the
37
airways that arises in the context of a complex interaction between genetic factors and
38
the evolving immune system of the infant and the environment to which it is exposed
39
[1,2]. Despite recent guidelines focus on asthma control, many asthmatic patients
40
remains poorly controlled in even under specialist care[3]. The outcomes might have
41
been improved by earlier diagnosis and better monitoring. Therefore, it is urgent to
42
find new biomarkers to measure and monitor the amount of inflammation within the
43
lungs of a patient with asthma and, as a result of better treatment of the disease.
44
Recently, several questions have been put forward on the role of chitinase and
45
chitinase-like proteins in inflammation and tissue remodeling in human disease.
46
The chitinase-like protein YKL-40, also called chitinase 3-like 1 (CHI3L1), is
47
produced at sites of inflammation in many cells and is secreted from macrophages and
48
smooth muscle cells [4]. YKL-40 binds to ubiquitously expressed chitin but is
49
deficient in chitinase activity. Previous studies have demonstrated that YKL-40 was
50
associated with pathologic conditions characterized by aberrant cell growth, tissue
51
inflammation and remodeling, such as several cancers, diabetes, atherosclerosis,
52
rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), liver
53
fibrosis, idiopathic pulmonary fibrosis and Crohn’s disease [4-14]. For some of these
54
diseases, the measurement of YKL-40 has been proven to be of both diagnostic and
55
prognostic value [5,7,14].
56
YKL-40 is synthesized in neutrophil precursors and stored in the specific
57
granules of neutrophils. YKL-40 secretion is stimulated by IL-6, IL-17,
58
IL-18-cytokines and released from neutrophils, vascular smooth muscle, macrophages,
59
chondrocytes and cancer cells[15]. YKL-40 has been shown to induce activation of
60
the mitogen-activated protein kinase pathway, nuclear factor-κB transcriptional
61
activity and protein kinase B (Akt) pathway in cell cultures like human colon cell
62
lines and human chondrocytes and synovial cells[16]. YKL-40 also potently
63
stimulates the growth of several types of human fibroblast derived from synovium,
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adult skin, and fetal lung[15]. Moreover, YKL-40 acts a chemoattractnat for modulate
65
vascular endothelial cell morphology by promoting the formation of branching
66
tubules[17]. A recent study by Tang et al. demonstrated that YKL-40 may be involved
67
in the inflammation of asthma by induction of IL-8 from epithelium, subsequently
68
contributing to BSMC proliferation and migration[18]. Collectively, YKL-40 has a
69
role in inflammation, pathological, fibrosis and tissue remodeling.
70
Chupp et al. demonstrated that YKL-40 was strongly unregulated in the
71
alveolar macrophages and subepithelial basement membrane of asthmatic patients,
72
and that serum YKL-40 level was elevated in asthmatic patients[10]. Duru et al.
73
showed that serum YKL-40 levels were higher in non-smoker asthma patients during
74
acute exacerbation than those of control individuals[19]. Kuepper et al. also indicated
75
that YKL-40 levels were predominantly increased at the site of allergen deposition in
76
response to allergen challenge [20]. A recent study suggested that serum YKL-40
77
levels were significantly elevated in patients with asthma compared with controls,
78
indicating that high levels of serum YKL-40 may be a biological characteristic of the
79
exacerbation of asthma[21].
80
Although previous studies have reported the elevated levels of YKL-40 in
81
asthmatic patients, the change in serum YKL-40 levels upon treatment in asthmatic
82
patients and its correlations with lung function and asthma control test (ACT) remain
83
unknown. Therefore, the aim of this study was conducted to estimate whether the
84
serum YKL-40 levels are decreased in patients with asthma after introduction of
85
appropriate treatment. Furthermore, the relationships between the serum YKL-40
86
level, lung function and ACT were also investigated in order to evaluate the clinical
87
significance of serum YKL-40 level during the course of the disease.
88
89
Methods
90
Study subjects
91
103 patients (47 women and 56 men, mean age 33.1 ± 0.9 years) were recruited
92
randomly from Outpatient Clinic, Pulmonary Department, Affiliated Hospital of
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Guangdong Medical College. Patients with no previous diagnosis of asthma
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94
presenting with respiratory symptoms were considered eligible for our study when
95
they were (a) cases who were older than 18 years of age, (b) able to perform
96
spirometry, (c) literate in Chinese. Asthma was diagnosed according to the GINA
97
guidelines based on a history of recurrent episodes of wheezing and chest tightness,
98
with or without cough, and impaired spirometry with reversibility in FEV1 of >12%
99
and 200ml after salbutamol administration or hyperresponsiveness to inhaled
100
methacholine[22]. Exclusion criteria were the following: current smoking or smoking
101
history of >5 pack years; oral corticosteroids or respiratory tract infection within the
102
preceding 4 weeks prior to enrolment; any chronic cardiopulmonary disease other
103
than asthma (including COPD); pregnant. We also obtained the specimens from
104
Clinical Research Center of Guangdong Medical College Tissue Bank. Although these
105
samples were not specifically collected for this study, our study was part of a project
106
that examined the molecular mechanism of inflammation in asthma. Bronchial-biopsy
107
specimens were collected from 5 normal subjects who were nonsmokers, 8 patients
108
with mild asthma, 8 patients with moderate asthma and 7 patients with severe asthma.
109
Their basic characteristics matched those in the present study participants.
110
The study protocol was approved by the Ethics of Research Committee of the
111
Medical College of Guangdong (PJ20130016) and was registered on the Chinese
112
Clinical Trial Database (ChiCTR-OCC-13003316). Written informed consent was
113
obtained from all participants.
114
115
Study design
116
At baseline visit, patients underwent a detailed clinical examination and completed
117
pulmonary function test, skin prick test (SPT), serum YKL-40 measurement, and ACT
118
questionnaire. Patients were treated with appropriate medication according to the
119
GINA guidelines [22] and were reevaluated 8 weeks later (visit 2). Serum YKL-40,
120
spirometry and ACT score were measured at follow-up visit. Intermittent asthmatic
121
patients received inhaled salbutamol 100-400 μg per day (Salbutamol Sulfate for
122
Inhalation, GlaxoSmithKline) as needed. Both mild to moderate and severe asthmatics
123
were treatment with low-to medium-dose budesonide (200-800 μg per day) delivered
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via turbuhalers (AstraZeneca AB) on a regular basis, plus a beta-2 agonist (either a
125
short-acting beta-2 agonist on an as-needed basis or a long-acting beta-2 agonist
126
(Formoterol Fumarate Powder For Inhalation, AstraZeneca AB) on a regular basis)
127
and/or a third controller (e.g., leukotriene antagonists or aminophylline). Adherence to
128
medical treatment was assessed using the Chinese version of Medication Adherence
129
Report Scale for Asthma (MARS-A10) items (Supplemental Table 1). MARS-A10
130
developed by Horne and collaborators is a brief self-measure that has demonstrated
131
good psychometric properties [23].
132
133
Pulmonary function tests
134
Spirometry was measured with standard spirometric techniques (Jaeger, Germany) at
135
least 6 hours after a patient’s most recent treatment with albuterol, according to
136
American Thoracic Society (ATS) guidelines[24]. Patients were divided into three
137
groups on the basis of severity of asthma, according to GINA [22]: (1) mild asthma
138
(FEV1 ≥80%); (2) moderate asthma (FEV1 60-79%); and (3) severe asthma (FEV1
139
<60%).
140
141
Skin prick test (SPT) and asthma control test (ACT)
142
Atopy was tested with the SPT. Twelve common animal and aeroallergens
143
(ALK-Abello, Horsholm, Denmark) were tested. The test was considered positive if
144
the wheal diameter of at least 3 mm. The ACT questionnaires administered to the
145
patients of this study had been formally translated into Chinese. Patients were
146
classified into three groups based on ACT scores [25]: completely controlled (ACT
147
score=25), partly controlled (ACT score range=20-24), and uncontrolled (ACT score
148
range=5-19). Asthma-related quality of life (QoL) was also measured by using the
149
Mini-Asthma QoL Questionnaire (MiniAQLQ). The mini AQLQ is a validated
150
15-item questionnaire, which assesses the quality of life specifically in relation to
151
asthma[26]. The questionnaire assesses three domains of asthma interference with
152
daily life (symptoms, environmental limitations, and emotions). The total score
153
obtained in the different domains was divided by 15 to obtain the score for each
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patient, with higher scores being indicative of better quality of life [26].
155
156
Serum YKL-40 and total serum IgE
157
Serum YKL-40 levels were measured using commercially available enzyme-linked
158
immunosorbent assay (ELISA) kit (Uscn Life Science Inc. Wuhan) according to the
159
manufacturer’s instructions. The minimum detection limit of the YKL-40 assay was
160
12.2 pg/ml. Total serum IgE levels were determined using a fluoroenzyme
161
immunoassay from asthmatic patients. The percentage of peripheral blood eosinophils
162
was detected using a routine blood test.
163
164
Immunohistochemistry
165
Deparaffinized sections (5 μm thick) were immersed in citrate antigen retrieval
166
solution, and preheated to 100°C for 20 minutes. After blocking endogenous
167
peroxidase with immunol staining blocking buffer for 60 min, the slides were then
168
incubated either with a rabbit polyclonal Ab against human YKL-40 (Uscn Life
169
Science Inc., Wuhan, China). The immunoreaction was visualized using
170
3,3-diaminobenzidine chromogen solution (DAB substrate kit, Abcam, Cambridge,
171
UK) and counterstained with hematoxylin according to the manufacturer’s
172
instructions. Quantitative measurements of YKL-40 positive cells in the bronchial
173
tissue were performed according to previous described [27]. Briefly, YKL-40-positive
174
and -negative cells were counted in each biopsy specimen separately in the intact
175
epithelium and the submucosa. Bronchial cells positive for the YKL-40 antibody were
176
expressed as a percentage of total cells.
177
178
Statistical analysis
179
Kolmogorov-Smirnov (KS) test was performed to examine normality of distribution.
180
YKL-40 levels were not normally distributed and the values were compared among
181
the study groups with the use of the nonparametric tests, including the Mann–Whitney
182
U–test and the Kruskal–Wallis test. The interrelationships between different
183
parameters were determined using Spearmans correlation. Receiver operating
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characteristic (ROC) curve analysis was performed to derive the optimal cutoff point
185
for YKL-40 as a predictor for increase in FEV1 and response to steroid treatment.
186
Data are presented as the mean ± SEM or n (%), unless otherwise stated. GraphPad
187
Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for the
188
analyses and graphs. Statistical significance was set at a p value < 0.05.
189
190
Results
191
Patient characteristics
192
A total of 103 patients with newly diagnosed asthma (56 men, 47 women, mean age
193
33.1 ± 0.9) were recruited for our study. They included 72 with mild asthma
194
(intermittent or mild), 20 with moderate asthma and 11 with severe asthma. Sixty-nine
195
patients (67.0%) had at least one positive SPT result. Sensitizations to house dust
196
mites and cockroaches were the most common. The mean baseline serum IgE, blood
197
eosinophils in WBC and body mass index (BMI) were 631.7 kU/L, 5.4% and 21.6
198
respectively. The characteristics of participants in our study were shown in Table 1.
199
During the follow-up visit, five patients were removed because they were
200
unwilling to complete the treatment and refused to continue participating. Finally, 98
201
subjects completed the study and were included in the data analyses. There were no
202
significant differences in baseline characteristics between patients who dropped out
203
and patients who completed the treatment (Supplemental table 2).
204
205
Baseline visit
206
At baseline visit, the mean %FEV1 was 84.1 ± 1.4%; 72 patients (69.9%) had FEV1
207
≥80%, 20 (19.4%) had FEV1 60-79%, and 11 (10.7%) had FEV1<60% (Table 1). The
208
mean ACT score was 20.6 ± 0.5; 26 patients (25.2%) were completely controlled, 52
209
(50.5%) were partly controlled, and 25 (24.3%) were uncontrolled (Table 1). Patients
210
with completely controlled asthma had statistically lower serum YKL-40 levels than
211
patients with partly controlled (p<0.001) and uncontrolled asthma (p< 0.001), but no
212
differences were detected in the serum YKL-40 levels between partially controlled
213
group and uncontrolled group (Fig. 1a).
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Serum YKL-40 levels for patients in the severe asthma group were higher than
215
those in the moderate asthma group (85.60 [56.2-94.3] ng/ml vs 76.00 [52.9-86.0]
216
ng/ml; p=0.024) and those in the mild asthma group (85.60 [56.2-94.3] ng/ml vs
217
63.05 [53.6-75.3] ng/ml; p<0.001). To confirm this result, bronchial biopsy specimens
218
specifically stained for YKL-40 in patients with asthma were further evaluated.
219
Quantification of lung YKL-40 expression revealed that YKL-40 expression was
220
higher in mild asthma compared to healthy controls (19.15% [14.60-20.75%] vs 11.70%
221
[9.01-13.85%]; p=0.006). Subgroup analysis revealed that YKL-40 expression was
222
higher in both severe and moderate asthma than mild asthma (41.0% [35.8-44.7%] vs
223
19.2% [14.6-20.8%]; p<0.001 and 29.2% [24.4-33.3%] vs 19.2% [14.6-20.8%];
224
p<0.001 respectively), with severe asthma exhibiting significantly higher YKL-40
225
levels than mild asthma (41.0% [35.8-44.7%] vs 29.2% [24.4-33.3%]; p=0.002)
226
(Figure 2). The serum YKL-40 level negatively correlated with %FEV1 (r=-0.37,
227
p<0.001) and ACT score (r=-0.26, p=0.007) at baseline visit (Fig.3).
228
229
Visit 2
230
At visit 2, the median serum YKL-40 level was 54.5 [46.4-58.4] ng/ml and
231
mean %FEV1 was 87.8 ± 0.9%; 85 patients (86.7%) had FEV1 ≥80%, 10 (10.2%) had
232
FEV1 60-79%, and 3 (3.1%) had FEV1 <60% (Table 1). The mean ACT score was
233
22.1 ± 0.3. Thirty-one patients (31.6%) were completely controlled, 60 (61.3%) were
234
partly controlled, and 7 (7.1%) were uncontrolled (Table 1).
235
statistically significant differences in serum YKL-40 values among patient groups
236
with different ACT score (Fig. 1b). No correlations between serum YKL-40
237
and %FEV1 or ACT score values were observed at visit 2 (r=-0.13, p=0.210 and r=
238
-0.07, p=0.511, respectively) (Fig. 4).
There were no
239
240
Baseline visit versus visit 2
241
As shown in Table 1, there were statistically significant differences in serum
242
YKL-40, %FEV1 value and ACT score between visit 1 and visit 2. Statistically
243
significant correlations were observed between changes (Δ) in YKL-40 and ΔFEV1
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(r=-0.28, p =0.006) (Fig.5a) as well as between ΔYKL-40 and ΔACT (r=-0.22,
245
p=0.037) (Fig. 5b).
246
level and an improvement in %FEV1 and in ACT score. The mean ΔYKL-40,
247
Δ %FEV1 and ΔACT values differed significantly between two medication groups
248
(Fig. 6).
249
response were assessed based on the low-dose administration of ICS (≤400 μg
250
budesonide per day) and medium to high-dose administration of ICS (>400 μg
251
budesonide per day). Patients receiving low-dose ICS have lower YKL-40 and
252
higher %FEV1 and ACT score at baseline versus those patients receiving medium to
253
high-dose ICS (Table 2). Patients with elevated levels of YKL-40 had significantly
254
greater corticosteroid use than patients with lower levels (table2).
Patients who received ICS showed a decrease in serum YKL-40
The outcomes and the relationship between treatment with ICS and
255
By ROC curve analysis, the value of the area under the ROC curve (AUC) for
256
FEV1 and steroid treatment were 0.76 (95%CI 0.67 to 0.86, p<0.001) and 0.75
257
(95%CI 0.63 to 0.85, p=0.002), respectively (Fig. 7).
258
Serum YKL-40 levels were positively correlated with blood eosinophils
259
(r=0.29, p=0.003) and were weakly positively correlated with total IgE levels (r=0.18,
260
p=0.058). No correlations between serum YKL-40 level, FEV1/FVC (%), age, SPT
261
and BMI and Mini-AQLQ were found during the study period.
262
263
Discussion
264
In this study, we further explored the potential role of serum YKL-40 measurement in
265
the management of asthma. The main findings of our study were as follows: (i)
266
elevated YKL-40 levels in asthma patients are associated with disease severity. Serum
267
YKL-40 levels were deceased in asthmatic patients after administration of appropriate
268
treatment; (ii) the serum YKL-40 correlated negatively with %FEV1 and ACT score
269
before asthma treatment; (iii) although these correlations were no longer observed
270
after treatment, changes in serum YKL-40, %FEV1 and ACT score continued to
271
correlate significantly; and (iv) patients who received ICS showed a significant
272
improvement in serum YKL-40, %FEV1 and ACT compared with patients without
273
ICS. Patients with elevated levels of YKL-40 had significantly greater corticosteroid
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use than patients with lower levels. These results provided evidence of YKL-40 level
275
is implicated in the pathophysiology of asthma.
276
At baseline serum YKL-40 levels in the partly controlled group and uncontrolled
277
group were significantly higher than those in the controlled group, but no differences
278
were detected in the serum YKL-40 levels between the partly controlled group and the
279
uncontrolled group. The reason for this is unclear, but may be explained by the
280
well-known individual variability in the perception of symptoms. Those who were
281
ACT remain uncontrolled (≤19)/YLK-40 levels below the median (≤75.5)
282
(Supplemental Fig. 1A) may have adequately addressed the atopic inflammation in
283
their airways after treatment, but continue to have symptom persistence for other
284
reasons, including co-morbidities (e.g., rhinitis, gastroesophageal reflux disease),
285
noneosinofilic asthma and masquerades of asthma. In comparison, those who were
286
ACT controlled (>19)/YLK-40 levels above the median (>75.5) (Supplemental Fig.
287
1B) were symptomatically controlled, but have evidence of persistent inflammation
288
and could be at risk for future asthma problems. Moreover, those who were ACT
289
uncontrolled (≤19)/YLK-40 levels above the median (>75.5) (Supplemental Fig. 1C)
290
may be allocated in the group of symptom controlled/high level of YKL-40 (>75.5)
291
asthmatics. This could be partly explained by a poor symptom-perception due to
292
adaptation to their level of disease. These data were consistent with previous reports
293
which suggested that asthma evaluation cannot be inferred from single measures of
294
ACT score[28-30]. GINA also recommend that questionnaires addressing the level of
295
asthma control need to be considered along with the objective measurements such as
296
lung function, inflammatory cytokines[22]. To reconcile this issue, we further
297
performed a separate analysis whereby the data was reanalyzed using the asthma
298
severity according to GINA. The data showed serum YKL-40 levels for patients in the
299
severe asthma group were higher than those in the moderate asthma group and those
300
in the mild asthma group. To confirm this result, bronchial biopsy specimens
301
specifically stained for YKL-40 in patients with asthma were further evaluated.
302
Quantification of lung YKL-40 expression revealed that YKL-40 expression was
303
higher in mild asthma compared to healthy controls. Subgroup analysis revealed that
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304
severe asthma exhibiting significantly higher YKL-40 levels than mild and moderate
305
asthma. These findings suggest that the ACT questionnaire may convey something
306
that inflammatory markers and spirometry cannot assess. However, asthma evaluation
307
control cannot be inferred from single measures of ACT score and should be
308
considered along with the objective measurements (e.g., biomarkers, lung function).
309
YKL-40 level is upregulated in human asthma and associated with disease severity,
310
suggesting that high levels of YKL-40 may be a biological characteristic of the
311
asthma severity.
312
Statistically significant correlations were observed between serum YKL-40 level
313
and %FEV1, as well as between serum YKL-40 level and ACT score. Specjalski et al.
314
showed that in Polish population, serum levels of YKL-40 were significantly higher in
315
patients with uncontrolled and party controlled asthma compared to controlled
316
asthmatic patients and healthy subjects[31]. Chupp et al. demonstrated that circulating
317
YKL-40 level was correlated with lung function, asthma severity, and the thickness of
318
the subepithelial basement membrane[10]. Additionally, a recent study by Tang et al.
319
showed that serum YKL-40 levels correlated positively with exacerbation attacks and
320
blood eosinophils, but correlated inversely with lung functions[21]. However, the
321
exact role of the serum YKL-40 level in asthma is still under debate. Specjalski et al.
322
showed that no relations were found between YKL-40 and asthma severity or total
323
serum IgE[31]. A recent study by Sohn et al. suggested that no significant associations
324
between CHI3L1 single nucleotide polymorphisms (SNPs) and asthma were observed
325
in an East Asian population [32]. The basis for these discrepancies may be due to
326
ethnic background and differences in CHI3L1 haplotype structures. A promoter single
327
nucleotide polymorphism in the gene encoding YKL-40, CHI3LI rs495028 (131C/G)
328
has associated with features of asthma and serum YKL-40 levels [33,34]. This
329
suggests that CHI3LI gene polymorphism may be influencing a reduction in YKL-40.
330
At visit 2, no correlations between serum YKL-40 and % FEV1 or ACT score
331
were observed. One explanation may be that a majority of patients in our study were
332
mild asthmatics and showed a response in all three measurements after administration
333
of appropriated therapy, minimizing the range of their distribution. However, the
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334
changes in serum YKL-40, %FEV1 and ACT score continued to correlate significantly.
335
Furthermore, serum YKL-40 level was declined in asthmatic patients after appropriate
336
treatment. One possible explanation for this result could be that ACT scores were
337
significantly improved in most patients who presented with a mild or stable condition
338
after treatment. This is consistent with previous studies by Chupp et al. and Tang et al.
339
showed that serum YKL-40 levels were higher in patients with asthma exacerbations
340
than in those in a stable condition[10]. In addition, our data demonstrated, as expected,
341
that patients with asthma receiving regular ICS therapy had a significant improvement
342
in serum YKL-40, %FEV1, and ACT. Previous study demonstrated that serum
343
YKL-40 in patients with active rheumatoid arthritis decreased rapidly during
344
prednisolone therapy, suggesting that steroids may have a direct effect on expression
345
of YKL-40[35]. However, it should be noted that patients were treated with ICS but
346
differences in dosing and compliance existed, and serum YKL-40 level may also be
347
influenced by CHI3LI gene polymorphism or other medications such as
348
bronchodilators. Patients with elevated levels of YKL-40 had significantly greater
349
corticosteroid use than patients with lower levels, suggesting that YKL-40 production
350
may be refractory to current asthma treatments and, therefore, may represent an
351
alternative therapeutic target for severe asthma[36].
352
There are some limitations that need to be considered. Asthmatic patients with
353
newly diagnosis asthma were enrolled in our study and most of them (69.9%, 72/103)
354
were mild asthma patients. Our results may not fully reflect general population of
355
asthmatic patients, especially patients with severe therapy-resistant asthma. The
356
observation period of our patients was relatively short. Additionally, we did not
357
consider to also using the Asthma Control Questionnaire (ACQ) under the assumption
358
that the ACQ has not been validated in the Chinese population and ACT is easier to
359
score and provides the same screening accuracy[37-39].
360
361
Conclusions
362
In summary, our current findings demonstrate that elevated YKL-40 levels in asthma
363
patients correlate with disease severity. Patients with elevated levels of YKL-40 had
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364
significantly greater corticosteroid use than patients with lower levels, suggesting that
365
high levels of serum YKL-40 may be refractory to current asthma treatments.
366
Prospective studies will be required to determine the role of YKL-40 in patients with
367
severe therapy-resistant asthma and whether the YKL-40 gene expression is under the
368
direct control of the corticosteroid.
369
370
Abbreviations
371
ACT: asthma control test; ICS: inhaled corticosteroids; CHI3L1: chitinase 3-like 1;
372
COPD: chronic obstructive pulmonary disease; GINA: Global Initiative for Asthma;
373
SPT: skin prick test; LABA: long acting β2-agonist; SABA: short acting β2-agonist;
374
ATS: American Thoracic Society; FEV1: forced expiratory volume in one second;
375
FVC: forced vital capacity; AQLQ: Asthma QoL Questionnaire.
376
377
Competing interests
378
None of the authors has any conflict of interest with the data published in this
379
manuscript. There are also non-financial competing interests to declare in relation to
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this manuscript.
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382
Authors’ Contributions
383
TWL: Performed experiments, analyzed data, wrote manuscript; YYL: performed
384
analyses and assisted in procuring human blood sample, reviewed manuscript; MC:
385
aided in data collection and analyses; DW and DML: performed ELISA and produced
386
graphs and tables; BW and DZC: designed the study and assisted in generating the
387
final manuscript. All authors have read and approved the final manuscript.
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Authors’ information
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1. Department of Respiratory and Critical Care Medicine, Affiliated Hospital, Institute
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of Respiratory Diseases, Guangdong Medicine College, Zhanjiang, China.
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2. Department of Respiratory Medicine, Affiliated Hospital of School of Medicine,
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Ningbo University, Ningbo, China.
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Acknowledgements
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This work was supported by grant of the Medical Scientific Research Project of
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Guangdong Province, China (No. A2012430) and Natural Science Foundation of
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Ningbo (No. 2012A610257).
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Figure legends
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Fig. 1 Classification of patients based on ACT score at baseline visit (a) and visit 2 (b).
560
Note: ***p < 0.0001; NS: not significant. Horizontal bars indicate mean values.
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Fig. 2 YKL-40 is unregulated in the bronchial tissue of patients with asthma. Specific
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staining for YKL-40 is brown, whereas nuclei are stained blue. Representative
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photomicrographs are present from A) a healthy individual and B) a patient with mild
565
asthma and C) a patient with moderate asthma, and D) a patient with severe asthma.
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YKL-40 positive and negative cells were counted in the epithelium and submucosa
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(expressed as a percentage of total cells). Data are presented as medians. E) YKL-40
568
is increased in both severe and moderate asthma compared with mild asthma, with
569
significant difference found between the two patient groups.
570
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Fig. 3 Correlation of serum YKL-40 levels to FEV1 (a) and ACT score (b) at baseline
572
visit. Note: FEV1, forced expiratory volume in one second; ACT, asthma control test.
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Fig. 4 Correlations of serum YKL-40 levels to (a) FEV1 and (b) ACT score at visit 2.
575
Note: FEV1, forced expiratory volume in one second; ACT, asthma control test.
576
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Fig. 5 Correlation of changes (Δ) in three parameters between the two visits.
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Statistically correlations were observed (a) between ΔYKL-40 and ΔFEV1; (b)
579
between ΔYKL-40 and ΔACT. Note: FEV1, forced expiratory volume in one second;
580
ACT, asthma control test.
581
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Fig. 6 Changes in asthma parameters in patient groups according to medication.
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Note: FEV1, forced expiratory volume in one second; ACT, asthma control test. ICS,
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inhaled corticosteroids; ICS (+), patients with ICS treatment; ICS (-), patients without
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ICS treatment.
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Fig.7 Receiver operating characteristic curve showing YKL-40 as a predictor for
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increase in FEV1 after treatment (black line) and predict response to steroid treatment
589
(red line). Sens, sensitivity; Spec, specificity; AUC, area under the ROC curve.
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Supplemental Fig. 1 Correlation of serum YKL-40 levels to ACT score at baseline
592
visit. A: ACT remain uncontrolled (≤19)/YLK-40 levels below the median (≤75.5); B:
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ACT controlled (>19)/YLK-40 levels above the median (>75.5); C: ACT uncontrolled
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(≤19)/YLK-40 levels above the median (>75.5).
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Table 1 Characteristic of the study participants*
Characteristic
Visit 1 (n = 103)
P-value
Visit 2† (n = 98)
Female, n (%)
47 (45.6)
45 (45.9)
NS
Age, yrs
33.1 ± 0.9
33.2 ± 0.9
NS
Body mass index
21.6 ± 0.3
21.4 ± 0.2
NS
Nonsmokers, n (%)
81 (78.6)
NA
Ex-smokers, n (%)
22 (21.4)
NA
Atopic by skin prick, n (%)
69 (67.0)
NA
Total IgE, kU/L
631.7 ± 94.1
568.4 ± 86.8
NS
Eosinophil in WBC%
5.4 ± 0.4
4.0 ± 0.3
< 0.001
FEV1/FVC, %
72.1 ± 0.6
74.9 ± 0.8
0.0026
FEV1, % predicted
84.1 ± 1.4
87.8 ± 0.9
0.0270
≥80%, n (%)
72 (69.9)
85 (86.7)
60-79%, n (%)
20 (19.4)
10 (10.2)
< 60%, n (%)
11 (10.7)
3 (3.1)
YKL-40, ng/ml
75.2 (55.8-86.8)
54.5 (46.4-58.4)
< 0.001
AQLQ
5.19 ± 0.11
5.35 ± 0.10
0.2937
ACT score
20.6 ± 0.5
22.1 ± 0.3
0.0101
25, n (%)
26 (25.2)
31 (31.6)
20-24, n (%)
52 (50.5)
60 (61.3)
5-19, n (%)
25 (24.3)
7 (7.1)
*Data are presented as mean ± SEM or n (%), except for YKL-40, median (IQR).
Note: FEV1, forced expiratory volume in one second; FVC, forced vital capacity;
AQLQ, Asthma Quality of Life Questionnaire; ACT, asthma control test; NS, not
significant; NA, not assessed.
† Five patients were dropped out.
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
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Table 2 Efficacy results in asthma patients at baseline and after treatment with budesonide
Variable
≤400 μg budesonide per day (n=50)
Baseline
>400 μg budesonide per day (n=31)
Visit 2
Baseline
Visit 2
(2.3) *
82.0 (1.8) ♯
§
FEV1, % predicted
90.8 (0.8)
91.2 (0.6)
71.6
ACT score
21.3 (0.7)
22.4 (0.5)
19.1 (0.6)
22.5 (0.4) ♯
59.1 (54.3-86.6)
47.5 (40.3-55.3) ♯
86.2 (56.4-96.7) *
55.7 (43.8-63.7) ♯
YKL-40, ng/ml
Data are presented as mean ± SEM, except for YKL-40, median (IQR).
♯p< 0.001 vs. baseline.
§
p< 0.001 vs. baseline (≤400 μg budesonide per day).
*p< 0.01 vs. baseline (≤400 μg budesonide per day).
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649
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651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
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667
668
669
670
671
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673
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676
677
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Supplemental Table 1 The Medication Adherence Report Scale for Asthma (MARS-A10)
Item
1. I only use my [Name of Medicine] when I need it
2. I only use it when I feel breathless
3. I decide to miss out a dose
4. I try to avoid using it
5. I forget to take it
6. I alter the dose
7. I stop taking it for a while
8. I use it as a reserve, if my other treatment doesn’t work
9. I use it before doing something which might make me breathless
10. I take it less than instructed
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
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Supplemental table 2 Characteristics of the study participants and participants
excluded*
Study participants
Participants excluded
Characteristic
P-value
(n = 98)
(n = 5)
Female, n (%)
45 (45.9)
2 (40.0)
0.63
Age, yrs
32.7 ± 0.8
34.1 ± 0.3
0.69
BMI
21.8 ± 0.3
21.5 ± 0.2
0.82
Total IgE, kU/L
633.5 ± 93.6
618.3 ± 36.1
0.97
Eosinophil in WBC%
5.3 ± 0.4
5.0 ± 0.2
0.86
FEV1/FVC, %
71.6 ± 0.6
72.8 ± 0.3
0.65
FEV1, % predicted
84.6 ± 1.4
83.9 ± 0.7
0.91
YKL-40, ng/ml
75.7 (56.1-85.3)
74.6 (52.4-83.7)
0.60
ACT score
20.1 ± 0.5
20.9 ± 0.2
0.72
AQLQ
5.11 ± 0.11
5.41 ± 0.09
0.54
*Data are presented as mean ± SEM or n (%), except for YKL-40, median (IQR).
Note: BMI, Body mass index; FEV1, forced expiratory volume in one second; FVC,
forced vital capacity; ACT, asthma control test; AQLQ, Asthma Quality of Life
Questionnaire.
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