The Turkish Journal of Pediatrics
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Regulatory T Cell Levels in Children with Asthma
Mutlu Yüksek1, Funda Erol1 , Deniz Güloğlu1, Figen Doğu1, Atilla Halil Elhan2, Emel Babacan1, Aydan İkincioğulları1
Departments of 1Pediatric Immunology and Allergy, and 2Biostatistics, Ankara University Faculty of Medicine, Ankara,
|Natural regulatory T (nTreg) cells are described by expression of a specific
transcription factor, FOXP3, on CD4+CD25+ cells. They play very important
roles in the suppression of allergic reactions and disorders. The aim of this
study was to obtain peripheral blood Treg levels among atopic asthmatic patients
before and during inhaled steroid treatment and to observe the effect of these
cells on the pathogenesis and treatment of asthma. CD4+CD25+FOXP3+ T
cells obtained from 20 healthy donors and from 16 atopic asthmatic patients
before and after inhaled glucocorticoid treatment were examined by flow
cytometer. The levels of CD4+CD25+ FOXP3+ Treg cells were higher in
asthmatic children who had been receiving inhaled glucocorticoids, when
compared to the control group and to the patients’ levels before treatment
(p<0.05). The present study suggests that at least one of the anti-inflammatory
effects of inhaled glucocorticoids in asthma depends upon induction of Treg
asthma, children, nTreg cells, glucocorticoid.
|Allergic asthma is a complex and heterogeneous
disease characterized by chronic inflammation
of the bronchial mucosa and airway
hyperresponsiveness (AHR). A majority of
patients with asthma have an atopic background.
The Th2 cell-derived cytokines interleukin
(IL)-4, IL-5, and IL-13 play a central role
in asthma and allergy-. The identification
of transcription factors controlling Th1 and
Th2 development further support the Th2
hypothesis since GATA3 is over-expressed
and T-bet under- expressed in the asthmatic
airway. However, recent advances in both
immunology and clinical phenotyping of asthma
have raised the possibility that the other
mechanisms may drive the pathology in some
patients with asthma or coexist with Th2 type
inflammation. Regulatory T (Treg) cells control
the development of autoimmune disease,
transplant rejection and allergic diseases and
also play a key role in peripheral tolerance.
Although two distinct subsets of Tregs have
been described to date, the best established are
CD4+CD25+ Tregs, which emerge from the
thymus: natural Tregs (nTreg). They constitute
5-10% of peripheral CD4+ T cells. These
cells also express variable numbers of surface
markers such as GITR, CTLA-4, CD152,
neuropilin, and CD45 RO. However, the
molecule that best defines the phenotype and
function of nTregs is a transcription factor,
namely the forkhead transcription factor 3
(FOXP3),. Mutation of FOXP3 results in
the depletion of CD4+CD25+ T cells and a
syndrome called IPEX (immunodysregulation,
polyendocrinopathy, enteropathy, X linked),.
The other category of Treg cells also derives
from the thymus but acquires its suppressive
activity in peripheral tissues: adaptive Tregs
(aTreg). Two subsets of aTreg cells are defined
according to cytokine expression: (i) Tr1
cells release IL-10 and (ii) Th3 cells release
transforming growth factor (TGF)-β. Neither
of these cells expresses FOXP3.
Corticosteroids bind to and activate the
intracytoplasmic glucocorticoid receptor, which
translocates into the nucleus and promotes antiinflammatory
genes. Inhaled glucocorticoids are the most effective and widely used
drugs for asthma. These drugs inhibit T cell
activation and reduce the expression of Th2
type cytokines, which may contribute to their
Thus, the aim of the present study was to
obtain peripheral blood Treg cell levels among
atopic asthmatic patients before and during
inhaled steroid treatment and to identify the
effect of these cells on the pathogenesis and
treatment of asthma.
|Material and Methods |
The study population consisted of 16 asthmatics
and 20 age-matched healthy children. An
informed consent was obtained from parents
of both patients and controls.
The asthmatic group included 3 patients with
moderate and 13 with mild asthma. All patients
fulfilled the classification criteria according
to the Global Initiative for Asthma (GINA)
guidelines. They were all symptomatic and
were not receiving inhaled steroids or any other
drugs at the beginning of the study. Skin prick
test was conducted with a panel of common
aeroallergen extracts in the presence of positive
and negative controls on the forearm. All our
patients were allergic to aeroallergens. Healthy
children did not have asthma or other atopic
or infectious diseases.
Ethylenediamine tetraacetic acid (EDTA)-
anticoagulated blood was derived during
the season in the morning for evaluation
of the CD4+CD25+FOXP3+ T cell ratio,
and subsequently, inhaled corticosteroids
(fluticasone propionate in 5 of 16 patients, 250
μg/day or budesonide in 11 of 16 patients, 400 μ/day) were started. No nasal steroids were
given for comorbid allergic rhinitis during the
study period. Approximately two months later
(X±SD: 58.25 ± 19.49 - mean 56.0 days), the
evaluation of the CD4+CD25+FOXP3+ T cell
ratio was repeated when the symptoms were
| ||Figure 1: Example of quantitative value of FOXP3 level and fluorescence intensity (FI) in the CD4+CD25high gate|
Three-color flow cytometry analysis was
performed on peripheral whole blood
collected in EDTA anticoagulant tubes. The
blood cells were stained with antiCD4-PC5
(Immunotech, Marseille, France), antiCD25-
FITC (Immunotech, Marseille, France) and
intracellular anti-FOXP3-PE (eBioscience, San
Diego, CA). Isotype-matched antibodies were
used as negative controls.
Staining with mAb anti-FOXP3 (Antihuman
FOXP3, eBioscience) was achieved according to
the protocol recommended by the manufacturer
(eBioscience) with IntraPrep Permeabilization
Reagent (Immunotech, Marseille, France).
Three subsets of CD4+ T cells were defined
according to CD25 staining: CD25-, CD25low
and CD25high. Cells expressing CD25high were
chosen and gated for the detection of FOXP3+
T cells (Fig. 1). Three-color cytometry was
performed with Cytomics FC500 (Beckman Coulter) by using the CXP software version
The Statistical Package for the Social Sciences
(SPSS) for Windows 11.5 was used for
statistical analysis. Student t test was used
to check age differences; Fisher’s exact test
was used for gender differences between the
two groups. Mann-Whitney U test was used
to test the difference in CD4+CD25+FOXP3+
T cell levels between the groups. Wilcoxon
signed ranks test was performed to compare
CD4+CD25+FOXP3+ T cell levels before and
during treatment in the asthmatic group. The
data are given as median (interquartile range)
and 25th-75th percentiles. A p value of 0.05 or
less was considered significant.
|There were no significant age or gender
differences between control and asthmatic
groups. Basic demographic features of patients
and the control group are given in Table I.
The levels of CD4+CD25+ T cells expressing
FOXP3 were found to be higher in asthmatic
children who had been receiving inhaled
glucocorticoids, when compared to the control
group and to the patients’ levels before
treatment (Fig. 2).
| ||Table I. Demographic Features of Asthmatic Patients and Controls|
|Asthma is the most frequent chronic disease
of childhood; however, the pathogenetic
mechanisms are not yet completely clarified.
Th2 cytokines play a central role in asthma by
secreting IL-4, IL-5 and IL-13. Th1 cells might
efficiently contribute to the effector phase in
some allergic diseases depending on the stage
of inflammation. Another subset of T cells
with immunosuppressive function, Treg cells,
inhibit both Th2 and Th1 responses. There
are two distinct phenotypes and mechanisms of
action of Treg cells, including thymus-derived
CD4+CD25+FOXP3+ naturally occurring
(nTreg) and peripherally occurring adaptive
Treg (Tr1 and TH3) cells. Adaptive Treg cells
do not express FOXP3,. The role of Tregs
in the pathogenesis of allergic diseases was
not defined until recently. Treg may block the
transition from the early activation stage to the
differentiated Th2 state, limit airway infiltrates,
and act to prevent inappropriate Th2 responses
to environmental allergens.
There are few but important reports
regarding the effect of glucocorticoids on
CD4+CD5+FOXP3+ T cells in asthmatic
patients. Karagiannidis et al. reported
that systemic glucocorticoids significantly
increased the FOXP3 mRNA expression in adult
patients with moderate and severe asthma,
correlating with IL-10 mRNA expression.
They showed that Tr1 cell induction depended
on FOXP3 expression. The increase in
CD4+CD5+FOXP3+ T cells with glucocorticoid
treatment is in accordance with the results
achieved in our study. It is strongly probable
that glucocorticoids induce both adaptive and
natural Treg cells simultaneously.
| ||Figure 2: Data points and median levels of FOXP3
expressing T cells and CD4+CD25+FOXP3+ T cell
levels of both control and asthmatic groups.|
Recently, Provoost et al. reported that FOXP3
protein expression within the CD4+CD25high
T cells was significantly lower in adult stable
asthmatic patients compared with healthy adult
control subjects. They also observed a tendency
for increased FOXP3 protein expression within
the CD4+CD25high T cells in glucocorticoidtreated
patients. In conclusion, they suggested
that treatment with inhaled glucocorticoids in
asthmatic patients might increase the FOXP3
protein expression. Because low levels of Tregs
in asthmatic subjects increased with inhaled
glucocorticoid treatment in our study, the
results of Provoost’s study strongly support
our findings. The present study could also be a
proof of their suggestion but it would be better
to determine the FOXP3 mRNA levels.
This is the first longitudinal follow-up study in
children with regard to CD4+CD5+FOXP3+
T cells and inhaled glucocorticoid treatment.
The study reveals that inhaled glucocorticoids
increase the CD4+CD5+FOXP3+ T cells in
atopic asthmatic children and demonstrates
that nTreg cells play important roles in the
pathogenesis and treatment responses in
Glucocorticoids used in either inhaled or
systemic form are the treatment of choice in
asthma and allergic diseases because of their
potent anti-inflammatory effects. Here, we have
shown that inhaled glucocorticoids increase
nTreg cell levels. This could be another antiinflammatory
mechanism of corticosteroids.
In conclusion, the results of this study suggest
that one of the anti-inflammatory effects of
inhaled corticosteroids is probably the induction
of nTreg cells. Therefore, the manipulation of
these cells is promising for the prevention/
treatment of allergic disorders.
This study was supported by Ankara University
Scientific Research Projects Coordination
Unit (BAP) with the project number
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