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CLINICAL RESEARCH
THE CHANGES OF SERUM CYTOKINES ACCORDING
TO THE LEVEL OF ASTHMA CONTROL
Ta Ba Thang1, Nguyen Giang Nam2, Nguyen Van Doan3, Dao Ngoc Bang1
1Respiratory center, Military Hospital 103, Military Medical University
2Thai Nguyen Medical University
3Bach Mai Hospital
ABSTRACT
Cytokines play an essential role in the pathogenesis related to the severity, monitor progress, and evaluate
ICS therapeutic response in asthma. Research objectives: Evaluate changes in serum levels of IL4,
IL5, IL13, and TNFα in patients with asthma control according to GINA after 3 months. Subjects and
methods: Research on 66 patients with asthma control by ICS and LABA according to GINA at Bach
Mai Hospital from February 2014 to August 2016. Serum levels of IL4, IL5, IL13, and TNFα are tested
by immunofluorescence assay. Results: The concentration of IL-5, IL-13 decreased significantly after
3 months of control. The concentration of TNFα decreased significantly after 1 month of control and
decreased the most after 3 months of control. Conclusion: The concentration of serum cytokines changes
significantly after asthma control.
* Keywords: Asthma; serum cytokine; Asthma control; GINA.
INTRODUCTION
Asthma management aims to achieve and maintain
overall asthma control by reducing the severity of
current symptoms and minimizing future risk [1].
Guidelines from the Global initiative for asthma (2)
and many countries recommend using a stepwise
approach to control asthma symptoms and reduce
risk based on steps of treatment according to
disease severity and other patient characteristics
(3). Chronic airway inflammation is the most
important pathogenesis mechanism of asthma.
Pre-inflammatory cytokines (interleukins, tumor
necrosis factor) reflect inflammatory response and
play the role to classify phenotypes (endotypes)
of asthma, helping of target treatment (specific
anti-inflammation), and prognosis of exacerbation
risks in the future. Control of inflammation is
key therapy in asthma management. Therefore,
an inhaled corticosteroid (ICS) is always the
first choice in asthma control (3). According to
current guidelines, the assessment of asthma
control is mainly based on clinical tools, such as
asthma control questionnaires, asthma control
tests, and pulmonary function. These tools have
the advantage of being easy to assess in clinical
practice. However, it is not able to assess changes
in the inflammatory process. So how do the
cytokines change, and are they related to asthma
control levels? Previous studies showed that there
was a relationship between some cytokines in
sputum or serum and levels of asthma control:
the correlation between IL-5, IL-8 in sputum and
Corresponding author: Ta Ba Thang (tabathang@yahoo.com)
Date received: 03/5/2021
Date accepted: 25/5/2021
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CLINICAL RESEARCH
asthma exacerbations (4); the increase of IL-5, IL-
8, IL-13 in serum relating to poor asthma control
(5, 6, 7); The “pro-inflammatory cytokine profile”
was not correlated with asthma control outcomes
(akiki). So is the change in serum cytokine a reliable
marker for assessing asthma control results?. This
study aims: To evaluate the change of serum IL-4,
IL-5, IL-13, and TNFα according to the levels of
asthma control.
SUBJECTS AND METHODS
1. Subjects
66 patients with a diagnosis of uncontrolled
moderate to severe persistent asthma at the
Asthma Outpatient Unit of the Clinical Allergy
and Immunology Center, Bach Mai Hospital from
February 2014 to August 2016.
* Selection criteria: (1) Patients aged ≥ 16 years, (2)
had received a diagnosis of asthma at least 1year
prior to study enrollment and were being managed
by the Asthma Outpatient Unit, (3) understand and
fill in the asthma control test (ACT). The diagnosis
of asthma was defined as the presence of
compatible clinical history and pulmonary function
tests demonstrating variable airflow obstruction by
means of bronchodilator responsiveness.
* Exclusion criteria: Patients had other respiratory
or cardiovascular diseases, cancer, diabetes,
chronic inflammatory diseases, tuberculosis,
smoked within 2 hours, or had respiratory tract
infection within the last 4 weeks.
2. Methods
* Study design: A prospective observational study.
Patients provided personal information, information
about their access to health care, current
symptoms, activity limitations due to asthma,
perceived asthma severity, perceived asthma
control, asthma medication usage, use of acute
health care services in the past year, monitoring
of their asthma, and they provided answers to
questions that probed their knowledge, attitudes,
and understanding of asthma and its treatment.
Patients were examined clinic, test of blood count,
pulmonary function test, serum cytokines before
treatment. Patients had started asthma treatment
with at least one of the following drugs: inhaled
corticosteroid (ICS), long-acting β 2 agonist
(LABA), ICS/LABA combination according to GINA
Guidlines (2013) [1]. At every month visit to the
outpatient clinic, patients were given guidance on
inhalation therapy and examined clinic (number
of exacerbations at night per week, number of
exacerbations per month, ACT, night waking
due to asthma, limitation of physical activities),
a test of blood count, pulmonary function test,
serum cytokines. The level of asthma control
was evaluated as “controlled,” “partly controlled,”
and “uncontrolled” based on a definition of GINA
guidlines (2013) [1]. Control treatment was
adjusted according to the level of asthma control
for every patient monthly. The study was approved
by the Ethics Council of Bach Mai Hospital. All
patients signed written informed consent.
Serum cytokine measurements: using a 9-plex
assay of R&D systems (USA) to quantify 9
cytokines (IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-
CSF, IFN-g, and TNF-a). The technique to quantify
cytokines was based on the fluorescence covalent
microbead immunosorbent assay. An aliquot of
the same human serum pool was run by duplicate
in each plate. In order to minimize inter-plate
variability, each serum cytokine value was divided
by the pool result obtained from the same plate and
expressed a as ratio. These ratios were included in
the statistical analyses.
3. Data analysis
Data management and statistical analysis were
performed by the SPSS version 12.0 software.
Data were expressed as “mean (standard deviation
[SD])”, percent (%), and 95% confidence interval
where appropriate. The Kruskal-Wallis and Mann-
Whitney U tests were used for independent groups
without normal distribution.
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CLINICAL RESEARCH
RESULTS
Table 1: General characteristics of patients.
General characteristics Results
Age: ( ± SD) (Min-max) (Years) 45.3 ± 16.74 (16 - 79)
Sex:
- Male (n, %)
- Female (n, %)
22 (33.33)
44 (66.67)
Atopic history (n, %) 46 (69.7)
BMI: ( ± SD) (kg/m2)
- Underweight (n, %)
- Normal (n, %)
- Overweight (n, %)
21.69 ± 2.81
2 (3.03)
51 (77.27)
13 (19.7)
Onset of asthma:
- Early (n, %)
- Late (n, %)
14 (21.21)
52 (78.79)
Number of exacerbation ≥ 1 time in the last year (n, %) 26 (39.4)
FEV1 ( ± SD) (% pred.) 48.25 ± 13.81
ICS treatment in the last year (n, %) 23 (34.8)
General characteristics of patients are summarized in Table 1: The average age of 45.3 ± 16.74 years,
with the higher rate of female (66.7% vs 33.3%), 69.7% of atopic history. The average BMI of patients was
21.69 ± 2.81 kg/m2, with 77.27% of patients having normal BMI and 19.7% of overweight patients. 78.79%
of patients had the late onset of asthma. 39.4% of patients have ≥ 1 time of exacerbation in the last year.
34.8% of patients were prescribed an ICS in the last year.
Table 2: Results of asthma control according to GINA guidline.
Control level
(n, %)
After 1 month
(1)
After 2 months
(2)
After 3 months
(3) p*
Well controlled 20 (30.3) 53 (80.3) 53 (80.3) p2.1 = 0.053
p3.1 = 0.004
p3.2 = 0.012
Partly controlled 32 (48.5) 11 (16.7) 6 (9.1)
Uncontrolled 14 (21.2) 2 (3.0) 7 (10.6)
p*: Fisher’ exact test
The results showed that 48.5% of patients had partly completed asthma while 30.3% of patients had well
controlled asthma and 21.2% of patients had uncontrolled asthma after 1 month. After 2 and 3 months the
rate of well controlled asthma was the highest (80.3%) while the rates of partly controlled and uncompleted
asthma decreased significantly in comparison with them in the 1st month (p < 0.05) (table 2).
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CLINICAL RESEARCH
Table 3: Changes of serum cytokine levels after asthma control.
Cytokine Median
(min - max) (pg/mL)
Pre-therapy
(1)
After 1 month
(2)
After 2 months
(3)
After 3 months
(4)
IL-4
1.01
(1.01 - 22.61)
1.01
(1.01 - 41.23)
1.01
(1.01 - 1.01)
1.01
(1.01 - 12.58)
p2.1 = 0.014; p3.1 = 0.008; p4.1 = 0.000; p3.2 = 0.000; p4.2 = 0.000; p4.3 = 0.000
IL-5
0.75
(0.75 - 31.21)
0.75
(0.075 - 6.46)
0.75
(0.075 - 0.75)
0.075
(0.075 - 17.45)
p2.1 = 0.000; p3.1 = 0.001; p4.1 = 0.000; p3.2= 0.013; p4.2 = 0.000; p4.3 = 0.000
IL-13
6.73
(6.73 - 656.69)
11.97
(1.6 - 3808.52)
11.97
(11.97 - 64.91)
1.6
(1.6 - 53.22)
p2.1 = 0.893; ; p3.1 = 0.159; p4.1 = 0.000; p3.2 = 0.396; p4.2 = 0.000; p4.3 = 0.000
TNFα
8.77
(0.5 - 21.12)
0.5
(1.0 - 262.87)
0.5
(0.5 - 17.21)
0.1
(0.1 - 32.31)
p2.1 = 0.000; p3.1 = 0.000; p4.1 = 0.000; p3.2 = 0.077; p4.2 = 0.000; p4.3 = 0.000
p: Wilcoxon signed-rank test
The average serum level of TNFα before the treatment was 8.77 pg/mL and after 1st, 2nd, 3rd month of
treatment were 0.5, 0.5, 0.1 pg/mL, reduced significantly versus before the treatment (p < 0.001). The
average serum levels of IL-5 and IL-13 before the treatment were 0.75 and 6.73 pg/mL and after 3th month
treatment were 0.075 and 1.6 pg/mL, reduced significantly versus before the treatment (p < 0.001). The
average serum levels of IL-4 after the treatment changed no significantly versus before treatment (table 3).
Table 4: Changes of serum cytokine levels after 1 month of control treatment.
Cytokine Median
(min - max) (pg/mL)
Uncontrolled
(n = 14) (1)
Partly controlled
(n = 32) (2)
Well controlled
(n = 20) (3) p*
IL-4 2.74
(1.01 - 41.23)
1.01
(1.01 - 35.12)
1.01
(1.01 - 7.64)
p2.1 = 0.446
p3.1 = 0.134
p3.2 = 0.366
p 0.444
IL-5 0.75
(0.075 - 5.41)
0.75
(0.075 - 6.46)
0.75
(0.075 - 0.75)
p2.1 = 0.498
p3.1 = 0.748
p3.2 = 0.614
p 0.808
IL-13 11.97
(1.6 - 3808.52)
19.47
(1.6 - 2958.57)
6.73
(1.6 - 101.64)
p2.1 = 0.533
p3.1 = 0.391
p3.2 = 0.05
p 0.168
TNFα 0.6
(0.1 - 162.87)
0.5
(0.1 - 29.31)
0.6
(0.1 - 13.39)
p2.1 = 0.748
p3.1 = 0.859
p3.2 = 0.793
p 0.936
p*: Mann-Whitney test p: Kruskal Wallis test
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CLINICAL RESEARCH
Table 5: Changes of serum cytokine levels after 2 months of control treatment.
Cytokine Median
(min - max) (pg/mL)
Uncontrolled
(n = 2) (1)
Partly controlled
(n = 11) (2)
Well controlled
(n = 53) (3) p*
IL-4 1.01
(1.01 - 1.01)
1.01
(1.01 - 1.01)
1.01
(1.01 - 1.01)
p 1.00
IL-5 0.75
(0.75 - 0.75)
0.75
(0.75 - 0.75)
0.75
(0.075 - 0.75)
p3.1 = 0.846
p3.2 = 0.649
p 0.995
IL-13 11.97
(11.97 - 11.97)
11.97
(11.97 - 23.94)
11.97
(11.97 - 64.91)
p2.1 = 0.529
p3.1 = 0.556
p3.2 = 0.821
p 0.923
TNFα 0.82
(0.5 - 1.13)
0.5
(0.5 - 5.0)
0.5
(0.5 - 17.21)
p2.1 = 0.422
p3.1 = 0.455
p3.2 = 0.790
p 0.839
p* Mann-Whitney test p Kruskal Wallis test
Table 6: Changes of serum cytokine levels after 3 months of control treatment.
Cytokine
± SD
(min - max) (pg/mL)
Uncontrolled
(n = 7) (1)
Partly controlled
(n = 6) (2)
Well controlled
(n = 53) (3) p*
IL-4 5.37 ± 1.13
(4.46 - 7.64)
5.61 ± 0.7
(4.46 0 6.58)
5.31 ± 1.54
(2.26 - 12.58)
p2.1 = 0.359
p3.1 = 0.962
p3.2 = 0.425
p0.709
IL-5 16.85 ± 44.36
(0.075 - 117.45)
2.84 ± 6.77
(0.075 0 16.65)
0.12 ± 0.25
(0.075 - 1.82)
p2.1 = 0.629
p3.1 = 0.039
p3.2 = 0.274
p0.571
IL-13 16.54 ± 20.28
(1.6 - 53.22)
1.93 ± 0.36
(1.6 - 2.26)
3.63 ± 8.0
(1.6 - 53.22)
p2.1 = 0.359
p3.1 = 0.159
p3.2 = 0.898
p 0.449
TNFα 11.64 ± 14.76
(0.1 - 32.31)
0.86 ± 1.85
(0.1 - 4.65)
0.14 ± 0.22
(0.1 - 1.62)
p2.1 = 0.229
p3.1 = 0.001
p3.2 = 0.274
p 0.221
p*: Mann-Whitney test p: Kruskal Wallis test