To the editor,
Asthma is a chronic airway inflammatory disease consisting of several
variable clinical presentations (phenotypes) and distinct
pathophysiological mechanisms (endotypes).1Chemoattractant receptor-homologous molecule expressed on Th2 cells
(CRTH2) is a type 2 receptor for prostaglandin D2 (PGD2), expressed on
Th2 and other various cell types including regulatory T cells
(Tregs).2 Tregs can suppress proliferation and
activation of effector type 2 inflammatory cells. On the other hand,
dysregulated Tregs have been implicated in developing chronic allergic
diseases.3 Among patients with allergic asthma, theFOXP3 gene expression and Treg suppressive function decrease
compared with healthy controls.4 GATA-3 expression in
Tregs may contribute to their Th2-like responses by switching Tregs to
Th2 cells under the type 2 milieu.5 Dysregulated Tregs
represent a Th2-like phenotype. That may be due to overexpression of
GATA3 and producing high amounts of interleukin (IL)-4 after local
exposure to PGD2.6 In this present study, we assessed
the presence of CRTH2+Tregs in peripheral blood of
patients with different inflammatory phenotypes of asthma. We found that
the frequency of CRTH2+Tregs was increased in patients
with asthma, particularly in those with concomitant allergy and this
increase correlated negatively with asthma control. Furthermore, we
discovered that the frequency of CRTH2+Tregs increased
during an acute asthma exacerbation. Finally, we demonstrated that Tregs
of allergic patients with asthma exhibited Th2-biased character.
We first studied the frequency of CRTH2+ and
CRTH2-Tregs
(CD3+CD4+CD25+CD127-)
and Th2
(CD3+CD4+CD25-CRTH2+)
in 120 patients with asthma compared with 30 healthy controls. For a
detailed description of the method, see the methods section in this
article’s supplementary data. The gating strategy is shown inFig S1A . The frequency of CRTH2+Tregs in the
asthma group, 0.09% [0.06-0.18], was significantly higher as
compared to the control group, 0.04% [0.03-0.06],p < 0.0001
(Fig 1A ). The frequencies of CRTH2-Tregs in
patients with asthma were significantly lower (Fig 1A ), whereas
Th2 cells did not differ between controls and asthmatics
(Fig S2A ). Next, we
compared the frequency of T cell subsets in patients with different
asthma phenotypes: eosinophilic allergic (EA), eosinophilic non-allergic
(EN), non-eosinophilic allergic (NA) and non-eosinophilic non-allergic
(NN)) patients and controls. The median of CRTH2+Tregs
in EA, NA, and NN was significantly higher than in controls (Fig
1B and S1B ). No significant difference was observed in the median of
CRTH2-Tregs and Th2 cells between asthma phenotypes
and control (Fig S2B ). Patients with allergic asthma had higher
frequencies of CRTH2+Tregs than those with
non-allergic asthma (Fig 1C ). Also, patients with controlled
asthma exhibited lower CRTH2+Tregs than those with
uncontrolled asthma (Fig 1C ). In this study, well-controlled
asthma was determined by a high asthma control test (ACT) score which
correlated with a low number of CRTH2+Tregs
(Fig 1D ). Besides, percentage of predicted peak expiratory flow
rate (PEFR) negatively correlated with CRTH2+Tregs
levels (Fig 1D ). We also observed that the frequency of
CRTH2+Tregs was associated with absolute eosinophil
counts and Th2 cells (Fig S2C ).
To explore the role of CRTH2+Tregs in an asthma
attack, we examined 30 asthma
donors during exacerbation in the absence of infection and 15 controls
(Fig S3A) . The level of CRTH2+Tregs was
significantly higher among the patients with asthma exacerbation, 0.13%
[0.1-0.22] than that of patients with controlled asthma, 0.09%
[0.08-0.11], p = 0.007 (Fig 1E and S3B ). After three
weeks of follow-up, the frequency of CRTH2+Tregs in
those patients during asthma control decreased significantly compared
with periodic asthma exacerbation, median difference 0.03%, p =
0.01 (Fig 1E ), whereas the differences were not significant
among patients with stable, controlled asthma.
Next, we analyzed the heterogeneity of T-cell phenotypes using
t-distributed stochastic neighbor embedding (tSNE) algorithm. T cell
populations could be classified based on clustering results, except
CRTH2+Tregs which grouped with Th2 cells (Fig
2A ). Of note, the numbers of CRTH2+Tregs were higher
among patients with allergic asthma than those with non-allergic
phenotypes and healthy individuals. To examine the relative expression
of Th2 genes in Tregs, we determined mRNA levels from purified Tregs of
patients with asthma and healthy controls. We observed statistically
higher expression of PTGDR2 mRNA in patients with allergic
phenotype compared with those with the non-allergic phenotypes and
healthy controls (Fig 2B ). Patients with asthma showed also
significantly reduced FOXP3 and a trend to higher expression ofGATA3 compared with controls (Fig 2C ). Accordingly, we
observed higher expression ofIL4 and IL13 in Tregs of patients with allergic asthma as
compared to healthy controls, but IL10 showed no significant
difference between the groups (Fig 2D ). These data indicate
that type-2 gene expression was elevated, whereas regulatory signature
was decreased in patients with asthma, particularly in those with
allergic phenotypes.
All patients during asthma exacerbation received systemic corticosteroid
treatment. We hypothesized that corticosteroids might affect the
frequency of the CRTH2+Tregs. To assess the efficacy
of dexamethasone and DP2 antagonist (ramatroban) in response to PGD2
engagement, we examined activated (CD69+)
CRTH2+Tregs. Reduced CD69 expression was observed in
the presence of dexamethasone with and without PGD2 compared with only
PGD2 stimulation (Fig 2E and S4A ). Under DP2 antagonist
treatment, the frequency of activated CRTH2+Tregs
decreased compared with the condition
with only PGD2 stimulation (Fig 2F and S4B ). These data
indicate that dexamethasone and DP2 antagonists could alleviate
CRTH2+Tregs activation.
Next, we determined whether CRTH2+Tregs might
influence the bronchial epithelial barrier. After the first hour of the
coculture, transepithelial electrical resistance (TER) was significantly
lower in the presence of CRTH2+Tregs with PGD2 than
CRTH2-Tregs (Fig 2G ). Noticeably, TER in the
presence of CRTH2+Tregs with or without PGD2
significantly decreased after 6, 24, 48, and 72 hours compared with the
control values (Fig 2G ). As shown in Fig S5 , TJ
protein was markedly disrupted in the HBEC-CRTH2+Tregs
cocultures compared with the absence of CRTH2+Tregs.
In
this study, CRTH2+Tregs stimulated with
PGD2 led to slightly increased levels of IL-4, IL-5 and
IL-13 compared with CRTH2+Tregs without of
PGD, but without
a statistically significant difference between both groups (Fig
2H ). Moreover, type 2 cytokines were produced by
CRTH2+Tregs more than CRTH2-Tregs
(Fig 2H ).
In the current study, we
investigated CRTH2+Tregs in a sufficient sample size.
However, the main limitation though is the lack of the broader and very
detailed investigation of the differences between
CRTH2+Tregs, CRTH2-Tregs and Th2
cells. Further studies will determine other surface markers,
transcription factors and cytokines which may lead to deeper
understanding of the importance of these populations in the steady state
and in allergic disease.
Here, we suggested that CRTH2+Tregs might be
implicated in the immunopathology of allergic asthma.
However,
CRTH2+Tregs are not very frequent and the suppressive
effect of CRTH2-Tregs may overwhelm these
dysfunctional Tregs. Currently, many novel antagonists for
PGD2 receptors have entered clinical trials. Information whether
CRTH2+Tregs might be a target for this new class of
drugs seems to be of particular interest. Additional validation of the
CRTH2+Tregs approach as a biomarker will be needed to
support clinical implementations.