Increased activated regulatory T cells proportion correlate with the severity of idiopathic pulmonary fibrosis
Hou et al. Respiratory Research
Increased activated regulatory T cells proportion correlate with the severity of idiopathic pulmonary fibrosis
Ziliang Hou 1 3
Qiao Ye 1 3
Meihua Qiu 1 3
Yu Hao 0 2
Junyan Han 0 2
Hui Zeng 0 2
0 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University , Jingshundongjie 8, Beijing 100015 , China
1 Department of Occupational Medicine and Toxicology, Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University , Worker's Stadium No.8, Chao-Yang District, Beijing 100020 , China
2 Beijing Key Laboratory of Emerging and Reemerging Infectious Diseases , Beijing , China
3 Department of Occupational Medicine and Toxicology, Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University , Worker's Stadium No.8, Chao-Yang District, Beijing 100020 , China
Background: Regulatory T cells (Tregs) are crucial in maintaining immune tolerance and immune homeostasis, but their role in idiopathic pulmonary fibrosis (IPF) is unclear. This study was designed to explore the role of Tregs in IPF. Methods: Percentages of Tregs and their subpopulations in peripheral blood (PB) and bronchoalveolar lavage (BAL) samples were determined by flow cytometry in 29 patients with IPF, 19 patients with primary Sjögren's syndrome-related interstitial pneumonia (pSS-IP), and 23 healthy controls (HCs). Results: In peripheral blood, no difference was found in CD4+CD25+Foxp3+ Treg percentages among patients with IPF, pSS-IP, or HCs. However, activated Treg (aTreg) fractions among CD4+ T cells increased significantly in IPF compared with pSS-IP or HCs. Being consistent with the result from the PB, aTreg fractions among CD4+ T cells in IPF also increased significantly compared with pSS-IP or HCs, accompanied by increased fraction III compared with HCs in BAL. IPF patients had lower levels of resting Tregs (rTregs) from the thymus than did HCs, whereas aTreg levels originating from the thymus did not significantly differ from HCs. Both rTregs and aTregs proliferated in IPF, with aTregs being more proliferative than rTregs. Both rTregs and aTregs significantly inhibited proliferation of CD4+ T lymphocytes in vitro. The percentage of aTregs was correlated negatively with predicted diffusing capacity values for carbon monoxide and positively with GAP index in IPF. Conclusions: Our study showed the imbalance between subpopulations of Tregs in IPF. Increased aTregs proportion in the peripheral blood correlated inversely with disease severity.
Idiopathic pulmonary fibrosis; Interstitial pneumonia; Primary Sjögren's syndrome; Regulatory T cells
Idiopathic pulmonary fibrosis (IPF) is a progressive and
fatal chronic lung fibrosis disease of unknown cause with
the histopathological features of usual interstitial
pneumonia (UIP) [
]. Accumulated evidence indicates that
IPF results from abnormal behaviour of the alveolar
epithelial cells that provoke migration, proliferation, and
activation of mesenchymal cells, causing the formation
of fibroblastic and myofibroblastic foci as well as the
destruction of the lung architecture [
Although it has been accepted that disturbed immune
homeostasis plays a significant role in the pathogenesis
and/or progression of IPF, the underlying mechanisms
remains unclear. IPF has a different fibrotic process than
connective tissue disease-related interstitial pneumonia
(CTD-IP). It has been proposed that the devastating
fibrotic response in IPF is driven by abnormally activated
alveolar epithelial cells [
] rather than a chronic
inflammatory process. Moreover, CTD-IP patients might benefit
from corticosteroids and/or immunosuppressive agents,
whereas IPF patients are unresponsive to
] and have few effective treatment options
beyond lung transplantation [
Regulatory T cells (Tregs), a subpopulation of T cells,
have immunosuppressive effects that maintain immune
tolerance and immune response homeostasis [
Accumulated data have shown that Tregs contribute to
maintaining immune homeostasis and are involved in a
number of respiratory diseases. However, the effects of
Tregs on lung fibrogenesis tend to conflict regarding
their pro-fibrotic or anti-fibrotic role in pulmonary
]. On one hand, Tregs could interfere with
upstream inflammatory events and indirectly decrease
the development of fibrosis through inhibition of
inflammatory and T helper cell responses [
]. On the other
hand, immunosuppressive Tregs are thought to possess
profibrotic functions by secreting the most potent
profibrotic cytokines, transforming growth factor (TGF)-β1
and platelet-derived growth factor (PDGF)-B [
Early studies showed that CD4+/CD25hi+/Foxp3+
Tregs were retained in the lungs of bleomycin-treated
CCR7−/− mice, which was consistent with an ameliorated
remodelling response to bleomycin-induced injury in the
]. The number and function of Tregs was
decreased in both peripheral blood (PB) and
bronchoalveolar lavage (BAL) samples from patients with IPF
compared to patients with CTD-IP and non-IPF lung diseases
]. In contrast, other studies reported higher circulating
Tregs in patients with rapidly progressive IPF [
Most of the studies used CD4+CD25+Foxp3+ to define
Tregs. However, recent research has demonstrated that
these classical-defined Tregs are heterogeneous and
separable into three functionally and phenotypically distinct
subpopulations: CD45RA+/CD25++ resting Tregs (rTregs)
and CD45RA−/CD25+++ activated Tregs (aTregs), both of
which are suppressive in vitro, and cytokine-secreting
CD45RA−/CD25++ T cells (Fr III), which are
]. Based on this novel definition, recent
studies have revealed the clinical relevance of
subpopulations of Tregs in autoimmune diseases [
infectious diseases [
]. Recently, we reported that patients
with chronic obstructive pulmonary disease (COPD) had
decreased rTreg and aTreg cells but significantly increased
Fr III cells in both PB and BAL compared with smokers
], which indicated a disturbed homeostasis of Treg
subpopulations in COPD. Here, by using the new
identification strategy for the Treg subpopulations, we found that
aTreg levels correlate with the disease severity of IPF, and
we explored the disturbed adaptive immune response
differences between COPD and CTD-IP.
We recruited 29 consecutive Chinese Han patients with
newly diagnosed IPF, 19 patients with primary Sjögren’s
syndrome-related interstitial pneumonia (pSS-IP), and
23 healthy controls (HCs) at Beijing Chao-Yang Hospital,
Capital Medical University, China (Table 1).
Diagnosis of IPF was based on the American Thoracic
Society (ATS)/European Respiratory Society (ERS)/Japanese
Respiratory Society (JRS)/Latin American Thoracic
Association (ALAT) statement [
]. Patients in the pSS-IP group
were diagnosed with pSS by the current guideline [
Their chest HRCTs showed a nonspecific interstitial
pneumonia pattern. Each patient was in a stable clinical and
functional state. Patients who presented with signs of heart
failure, acute pulmonary infection or pulmonary
thromboembolism were excluded, as were those who were receiving
treatment with corticosteroids and/or immunosuppressants
at the time of our study. Healthy controls are volunteers
recruited whose age matched with case groups, without
evidence of age-related arterial hypertension, diabetes,
cardiovascular disorders and cerebral vascular disease. The study
protocol (No. 81370159) was approved by the Ethics
Committee of Beijing Chao-Yang Hospital, and all participants
provided written informed consent.
Peripheral blood (PB) samples were obtained from each
subject and were processed to obtain peripheral blood
mononuclear cells (PBMCs) by density centrifugation.
BAL was performed and processed as previously
] in 7 patients with IPF and 12 patients
with pSS-IP. Nine subjects underwent routine healthy
examinations, received diagnostic bronchoscopy and
showed normal BAL cytology [
]. BAL and PB
samples were processed immediately after collection.
BAL cell differentials of the study groups are shown
in Table 2.
Flow cytometry analysis
Fresh PBMCs and BAL cells were stained with the
following antibodies: CD45RA-FITC, CD45RA-APC, CD25-PE,
CD25-PerCP-Cy5.5, CD4-FITC, CD4-PE-cy7, CD4-APC,
CD31-PE, and matched isotypic controls and incubated
medium (RPMI 1640 with 10% fetal calf serum).
CFSElabeled cells was assessed by flow cytometry.
for 30 min at 4 °C in dark room. For intracellular
staining, cells were fixed, permeabilized, and stained
with Foxp3-PE and Ki-67-FITC, according to the
manufacturer’s instructions. All antibodies were
purchased from BD Biosciences or Pharmigen (San Jose,
CA) and e-Bioscience (San Diego, CA). Data
acquisition and analysis were performed with a FACSCalibur,
which was equipped with CellQuest Pro software (BD
Biosciences, San Jose, CA). Approximate 105 cells
were acquired for subsequent data analysis.
Cell sorting and proliferation assay
For functional assays, CD25+ T cells were firstly isolated
by positive selection of PBMCs (obtained from 30 ml
whole blood) labeled with magnetic-bead conjugated
antihuman CD25 mAbs using MACS MultiSort Kit according
to manufacturer’s instructions (Miltenyi) from IPF
patients. Purified CD25+ T cells were stained with CD4,
CD25, and CD45RA antibodies, and then sorted into CD4
+CD25++CD45RA+ cells (rTregs, Fr I, 1 × 104), CD4
+CD25+++CD45RA− cells (aTregs, Fr II, 1 × 104), and CD4
+CD25++CD45RA− cells (Fr III, 1 × 104) using a FACS
Aria II flow cytometer (Becton Dickinson). The purity of
the Treg subsets was more than 95%. The CD4+CD25−
cells (responder T cells, 2 × 104) from healthy donors
(10 ml whole blood) can also be isolated using
magneticbeads conjugated anti-human CD25 mAbs and
antihuman CD4 mAbs.
CD4+CD25− responder cells (2 × 104) from healthy
donors were labeled with 1 μM CFSE (Invitrogen, OR,
USA) and were then cocultured with (1 × 104)
unlabeled, sorted rTreg, aTreg and Fr III cells at a 1:2
Treg subpopulations/CD4+CD25− responder cell ratio in
anti-CD3 (5 μg/ml OKT3 mAb; eBioscience) coated
plates in the presence of soluble anti-CD28 (5 μg/ml;
eBioscience) for 72 h at 37 °C and 5% CO2 in complete
The gender-age-physiology (GAP) index
The multidimensional GAP index is a simple and
reliable tool for disease severity stratification in IPF. We
calculated the GAP score for every IPF patient according
to the method reported by Ley et al. [
All analyses were performed with SPSS for Windows
V16.0 (Chicago, Illinois, USA). Values are presented as
the mean ± standard (SD) or as the median and IQR
when appropriate. Groups were compared using analysis
of variance, Student’s t-test, Wilcoxon rank-sum test, or
a chi-square test as appropriate. Correlations were
assessed using a Pearson correlation test or Spearman’s
rank test. A p value <0.05 was considered significant.
The characteristics of 29 patients with IPF, 19 with
pSSIP and 23 HCs are summarized in Table 1. Most (93%)
of the IPF patients had a history of smoking, but none
were current smokers. Compared with the IPF group,
more patients with pSS-IP were female (p < 0.001).
Pulmonary function values did not significantly differ
between the IPF and pSS-IP groups.
Increased frequencies of circulating aTregs in patients with IPF
We first investigated the percentages of circulating Tregs
classically defined as CD4+CD25++ in different groups.
Compared with HCs, patients with IPF or pSS-IP did not
have significant differences in the percentages of CD4
+CD25++ cells (HCs: 7.22 ± 1.59%; IPF: 7.70 ± 2.68%;
pSSIP: 6.67 ± 1.31%; Fig. 1c).
Since it has been shown that the degree of Foxp3
expression is proportional to CD25 expression in
circulating CD4+ T cells, we divided CD4+CD25++ T cells into
three subpopulations: rTregs (CD45RA+/CD25++, Fr I),
aTregs (CD45RA−/CD25+++, Fr II) and the
cytokinesecreting subpopulation (CD45RA−/CD25++, Fr III), as
described previously (Fig. 1a–d) [
13, 17, 19
]. We found
that the patients with IPF or pSS-IP and HCs showed
comparable percentages of Fr III cells (Fig. 1e). However,
the patients with IPF and patients with pSS-IP exhibited
significantly decreased percentages of rTreg fractions
(0.73 ± 0.52% and 0.84 ± 0.31%, respectively) when
compared with HCs (1.49 ± 0.62%, p < 0.001; Fig. 1f ). We
also observed comparable percentages of circulating
aTregs in patients with pSS-IP and healthy controls
(p = 0.952). Strikingly, compared with patients with
pSSIP and HCs, patients with IPF had an increase in the
aTreg fraction (1.29 ± 0.61%; p = 0.003 and p = 0.028,
respectively; Fig. 1g).
Next, in order to explore the immune functions of
Treg subpopulations in patients with IPF, we performed
an in vitro CFSE proliferation assay. Consistent with our
previous study [
], we found that both rTregs and
aTregs exhibited potent suppressive effects on responder
cells, while Fr III cells showed a mild suppressive
capacity compared with responders alone (Fig. 1h).
Impaired thymic output and enhanced proliferation account for imbalance of Treg subsets in IPF patients
To further investigate the imbalance of Treg subsets in
patients with IPF, we assessed CD31 expression in Treg
subpopulations, which is a marker for recent thymic
] and enable the discrimination of recent
thymic emigrant Treg cells from peripherally expanded
naive Treg cells [
]. A decrease in the CD31+ fraction
of rTreg cells was observed in IPF patients when
compared to age-matched HCs (27.79 ± 13.81% vs
45.59 ± 13.44%, p = 0.005; Fig. 2a), suggesting that
thymic production of Treg cells was affected in IPF
patients and the impaired thymic output contributed to
the decrease of rTreg cells. The maintenance of aTregs
can be achieved by extensive turnover of the existing
aTreg cells or by peripheral conversion of rTreg or
nonTreg cells into aTreg cells. However, comparable
percentages of the CD31+ fraction (mean <10%) were
observed in aTregs from IPF patients and HCs (Fig. 2b),
which might indicate only a small number of aTreg cells
were converted from rTreg cells and contributed partly
to the maintenance of aTreg cells pool. In the present
study, we assessed the proliferative activity of Treg
subsets by their expression of Ki-67, a nuclear protein
that is expressed at a higher level in proliferating cells
]. Compared with HCs, IPF patients displayed a slight
increase in the percentages of Ki-67+ cells in rTreg
(0.72 ± 0.34% vs 3.98 ± 2.44%, p = 0.004; Fig. 2c) but a
more dramatic increase in aTreg subsets (4.54 ± 2.86%
vs 35.20 ± 12.07%, p < 0.001; Fig. 2d), which indicates
that aTregs in IPF patients are hyper-proliferative.
Increased proportions of circulating aTregs correlated with disease severity in IPF patients
We first investigated whether frequencies correlate with
pulmonary function parameters, including forced vital
capacity (FVC) and DLCO that are used to predict IPF
severity. As shown in Fig. 3, the percentages of aTregs in
CD4+ T cells were negatively correlated with DLCO
predicted values (r = −0.475, p = 0.016; Fig. 3a) but not with
FVC % predicted values (r = −0.257, p = 0.225; Fig. 3b)
in IPF patients. However, we did not notice correlation
between the percentage of aTregs and predicted DLCO
% in pSS-IP patients (data not shown). As reported by
Ley et al. [
], GAP index and staging system has been
used as a quick and simple screening method for
informing prognosis in patients with IPF. We further
noticed that the percentage of aTreg cells was positively
correlated with GAP index in IPF patients (r = 0.488,
p = 0.018; Fig. 3c).
Increased proportions of aTregs in BAL from patients with IPF
To further test whether disturbed homeostasis among
Treg subpopulations also occurred in the lungs, we
assessed the proportions of Treg subpopulations in BAL
samples from 7 IPF patients, 12 pSS-IP patients and 9
HCs. In line with our previous study [
], the majority of
Tregs in BAL were CD45RA− (Fig. 4a and c), which
indicated that rTreg might only play limited roles in the local
immunity of these patients.We used the combination of
CD45RA and Foxp3 to separate Treg cells in BAL into
two subsets, CD45RA−Foxp3low and CD45RA−Foxp3high
(Fig. 4a and c). Differing from the results from circulating
Fr III, both IPF patients and pSS-IP patients showed an
increase in the percentages of Fr III fractions in BAL
compared to HCs (5.86 ± 2.13%; p = 0.035 and 0.030,
respectively) (Fig. 4d). More importantly, the results of aTregs
from BAL were consistent with those from the PB. The
patients with IPF showed significantly increased
percentages of aTregs in BAL (1.73 ± 0.55%) than patients with
pSS-IP (0.57 ± 0.40%, p = 0.002) and HCs (0.48 ± 0.21%,
p = 0.002) (Fig. 4e).
IPF is a chronic, progressive fibroproliferative interstitial
pneumonia of unknown aetiology. Various studies have
clarified the crucial roles of Tregs in many diseases, but
contradictory results have been obtained from both
murine models and IPF patients. Here, by using the new
definition of Treg subpopulations, we showed that,
compared with HCs and the patients with pSS-IP,
patients with IPF had larger fractions of circulating aTregs,
which was negatively correlated with DLCO predicted
values and positively correlated with GAP index.
One of the most important findings of the present
study is exploring a characterized disturbance of Treg
subpopulations in patients with IPF. Consistent with the
protective effects in pSS and other autoimmune diseases
], a slight decrease in CD4+/CD25+ Treg cells was
observed in patients with pSS. Here, we also observed a
significant decrease in the percentages of rTregs in
patients with pSS-IP, while the percentages of aTregs
remained comparable with the HCs. This finding
indicated that inflammatory responses are related to a loss
of rTregs in these patients. Similarly, IPF patients also had
decreased percentages of rTregs. However, a dramatic
increase in the percentages of aTregs was noticed. More
importantly, a more dramatic increase in aTreg frequencies
was observed in BAL samples of the patients with IPF.
Thus, the disturbed homeostasis patterns between the IPF
and pSS-IP patients were quite different. An increase in
aTregs is a hallmark of disturbed immune homeostasis in
IPF patients and might compensate for the loss of the
More importantly, although IPF patients display
distinct defects in Treg subpopulations compared to
patients with pSS-IP, a negative correlation was also seen
between circulating aTregs and DLCO in IPF patients.
Decreased rTregs and increased aTregs both showed
detrimental effects in patients with pSS-IP and with IPF,
respectively, which indicated that varying mechanisms
contribute to fibrosis formation in these two diseases. In
addition to a decreased number of Treg cells in pSS
patients, in vitro functional assays showed decreased
suppressive activity of CD4+/CD25+ Treg cells [
defects in Treg cells might result in an overwhelming
inflammatory response and subsequently cause tissue
destruction in the lungs.
By contrast, in addition to an increase in aTreg cells, our
in vitro functional analysis demonstrates that rTregs from
IPF patients are functionally immunosuppressive. Our
study showed that in addition to increased percentages of
aTregs, inflammatory BAL Fr III cells were also increased.
Despite the elevation of these two subpopulations, the
aTreg/Fr III ratio in IPF patients was higher than in pSS-IP
patients but comparable to that in HCs. From the above
data, immune homeostasis seems to remain intact in IPF
patients, although inflammatory and anti-inflammatory
mechanisms were activated. This might explain why IPF
patients are unresponsive to corticosteroids and/or
immunosuppressive agents, whereas pSS-IP patients might
benefit from them.
The disturbed homeostasis among Treg subpopulations
in IPF may reflect cell derivation. All T cells, including
these subpopulations, are derived from progenitors in the
bone marrow and differentiate in the thymus [
various stimulations, rTregs can upregulate Foxp3
expression, differentiate to aTregs and continue to proliferate
]. rTregs may represent the de novo generation of
thymic lymphocytes, that the assessment of rTregs is
possibly used to evaluate thymic Treg cell production.
Howerver, rTregs can proliferate after thymic export while
retaining their naive phenotype [
]. The surface
expression of CD31 has been used as a direct marker of
thymic output and enabled the discrimination of recent
thymic emigrant Treg cells from peripherally expanded
rTreg cells [
]. We found that the percentage of CD31+
rTregs in IPF patients was significantly lower than in
healthy controls, whereas CD31+ aTregs levels did not
significantly differ between IPF patients and healthy controls.
These results suggest an impaired thymic output of Tregs
in IPF patients. The extensive turnover of the existing
aTreg cells or by peripheral conversion of rTreg cells or
non-Treg cells into aTreg cells have been proved to
maintain the aTregs pool. The percentages of the CD31+
fraction in aTregs were low and in comparable level in IPF
patients and HCs, indicating IPF might not affect
conversion of rTregs to aTregs and contribute partly to the
maintenance of aTreg cells pool. Actually, rTregs and
aTregs represent essentially distinct populations from the
genomic standpoint [
].In present study, we assessed the
proliferative activity of individual Treg subsets by their
expression of Ki-67 and found significantly increased
percentage of Ki-67+ fractions in aTregs and rTregs in IPF
patients compared with healthy controls. Thus,
hyperproliferation at least partly contributed to the high levels
of circulating aTregs in IPF patients. These observations
hint that a shift in the homeostatic composition of Treg
subsets related to an impaired thymic-dependent de novo
generation of recent thymic emigrant rTreg cells with a
compensatory expansion of aTreg cells may contribute to
imbalance of Treg subsets in IPF patients. What is
noteworthy is that aTregs represent a highly differentiated
population, characterized by short telomeres, inability to
upregulate telomerase and susceptibility to apoptosis [
Therefore, aTregs have limited capacity for self-renewal,
suggesting that it is unlikely that the aTregs pool is
maintained through unintermittent turnover of existing aTregs.
Some studies have suggested that aTregs can develop from
Foxp3−/CD4+ non-Tregs [
] or from Fr III cells [
]. It is
of interest to investigate whether there is an alternative
pathway of generation of aTregs and how different
pathways contribute to the maintenance of the aTregs pool in
Of note, it has been shown that Tregs can contribute
to lung fibrosis by stimulating fibroblasts through the
secretion of PDGF-B under non-inflammatory
conditions and regulate detrimental T-cell activity during
inflammation-related fibrosis [
]. Moreover, activated
Tregs are capable of producing the potent pro-fibrotic
cytokine TGF-β1 . Thus, it is not surprising that the
percentage of circulating aTregs among CD4+ T cells in
IPF patients was inversely correlated with DLCO
predicted values. The multidimensional GAP (gender [G],
age [A], and 2 lung physiology variables [P] [FVC and
DLCO]) index and staging system is a simple method
for informing prognosis, helping guide management
decisions in patients with IPF [
]. Furthermore, the
percentage of aTreg cells was positively correlated with
GAP index in IPF patients.
Our study has some limitations that deserve comment.
One limitation of this study is that the potential role of
aTreg cells in chronic fibrotic lungs was not fully
declared. Further research work is warranted to disclose
whether the cells are per se that induce fibrogenesis or it
is the pro-fibrotic microenvironment that shifts them
towards a pro-fibrotic phenotype. Secondly, despite the
appreciable effort of obtaining BAL samples, the results
from the small size of the patients obtaining BAL may
In conclusion, our study provided further evidence for
the role of adaptive immunity in the pathogenesis of IPF
and showed an imbalance among subpopulations of
Tregs in IPF. Importantly, the increased aTregs in the
PB correlate inversely with disease severity. Our study
may suggest Tregs as potential future therapeutic targets
by restoring their homeostasis. Treg subpopulations may
be promising prognostic factors for IPF as well.
ALAT: Latin American Thoracic Association; aTregs: Activated Tregs;
ATS: American Thoracic Society; BAL: Bronchoalveolar lavage;
CFSE: Carboxyfluorescein diacetatesuccinimidyl ester; COPD: Chronic
obstructive pulmonary disease; CTD-IP: Connective tissue disease-related
interstitial pneumonia; DLCO: Diffusing capacity of the lung for carbon
monoxide; ERS: European Respiratory Society; FEV1: Forced expiratory volume
in the first second; FVC: Forced vital capacity; HCs: Healthy controls;
IPF: Idiopathic pulmonary fibrosis; JRS: Japanese Respiratory Society;
PaO2: Partial pressure of arterial oxygen; PB: Peripheral blood;
PBMCs: Peripheral blood mononuclear cells; PBS: Phosphate-buffered saline;
PDGF: Platelet-derived growth factor; pSS-IP: Primary Sjögren’s
syndromerelated interstitial pneumonia; rTregs: Resting Tregs; SSC: Side scatter;
TGF: Transforming growth factor; TLC: Total lung capacity; Tregs: Regulatory
T cells; UIP: usual Interstitial pneumonia
We thank all the patients who agreed to participate in the study and the
staff of the Lung Function Lab and Bronchoscope Lab at Beijing Chao-Yang
Hospital for their assistance.
This work was supported by the National Natural Science Foundation of
China (No. 81370159) and the Beijing Natural Science Foundation of China
Availability of data and materials
Data and materials are available on reasonable request by contacting the
first author Ziliang Hou ().
ZLH performed all data collection, collected and processed samples, and wrote
the manuscript. MHQ and YH performed laboratory-based assays. JYH was
responsible for analysing the data. HZ contributed as a primary investigator and
was responsible for designing the study and analysing the data. QY contributed
as a primary investigator and was responsible for designing the study, recruiting
the patients and writing the manuscript. All authors read and approved the final
Ethics approval and consent to participate
Research ethics approval was obtained from Ethics Committee of Beijing
Chao-Yang Hospital, Capital Medical University. All subjects provided written
Consent for publication
The authors declare that they have no conflict of interests.
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