Practical Synthesis of Phosphinic Amides/Phosphoramidates through Catalytic Oxidative Coupling of Amines and P(O)?H Compounds

Article abstract of DOI:10.1002/chem.201904237

Herein, we report a highly efficient ZnI₂-triggered oxidative cross-coupling reaction of P(O)?H compounds and amines. This operationally simple protocol provides unprecedented generic access to phosphinic amides/phosphoramidate derivatives in good yields and short reaction time. Besides, the reaction proceeds under mild conditions, which avoids the use of hazardous reagents, and is applicable to scale-up syntheses as well as late-stage functionalization of drug molecules. The stereospecific coupling is also achieved from readily available optically enriched P(O)?H compounds.

Full text of DOI:10.1002/chem.201904237

I M M U N O L OG Y O R I G I N A L A R T I C L E
Regulatory T-cells promote pulmonary repair by modulating T
helper cell immune responses in lipopolysaccharide-induced acute
respiratory distress syndrome
Wen Tan,
Chaoji Zhang,
Summary
Jianzhou Liu and Qi Miao
Acute respiratory distress syndrome (ARDS) induces a strong local infil-
tration of regulatory T-cells (Tregs) in the lungs. However, at present,
there remains a lack of adequate evidence showing the direct effect of
Tregs on pulmonary repair and the related mechanisms of ARDS. There-
fore, in this project, we studied the impact of Tregs on lipopolysaccharide
(LPS)-induced ARDS and pulmonary inflammation. Surprisingly, we
found that depletion of Tregs by injection of PC61 anti-CD25 antibody
not only interfered with the inflammation resolution, such as inhibited
total cell infiltration into the alveolar space, downregulated neutrophils,
upregulated macrophages, but also impaired pulmonary epithelium and
endothelial cell proliferation. Consistent with the attenuation of pul-
monary repair, we found that the Th1 and Th17 immune responses were
also impaired in Treg-depleted mice, suggesting that the presence of Tregs
is vital for tissue repair, as Tregs modulate and promote the Th immune
response in LPS-induced pulmonary inflammation.
Department of Cardiac Surgery, Peking Union
Medical College Hospital, Chinese Academy of
Medical Sciences and Peking Union Medical
College, Beijing, China
doi:10.1111/imm.13060
Received 8 November 2018; revised 7 March
2019; accepted 21 March 2019.
Correspondence: Qi Miao, Peking Union
Medical College Hospital (East), No. 1
Shuaifuyuan Wangfujing Dongcheng,
District, Beijing 100730, China.
Email: miaoqipumc@hotmail.com
Senior author: Qi Miao
Keywords: acute respiratory distress syndrome; regulatory T-cells; T helper
cell immune responses.
the alveolar endothelial–epithelial barrier contribute to
the development of ARDS. While neutrophils, monocytes
Introduction
Acute respiratory distress syndrome (ARDS) is a syn-
drome of respiratory failure. Although ARDS can be trig-
gered by a variety of predisposing conditions, including
pneumonia, aspiration, sepsis, trauma, blood transfusion
and pancreatitis, the most common cause of ARDS is
bacterial infection. This syndrome is associated with high
mortality and poor prognosis. With in-hospital mortality
ranging from 30% to 70%, on average, 86ꢀ2 out of
100 000 people acquire ARDS, and 75 000 deaths each
year are attributed to ARDS in the USA.1 Although exten-
sive investigations have been conducted, the underlying
mechanisms remain elusive. Thus, it is necessary to fur-
ther explore the mechanism of ARDS and find a new
therapeutic approach to enhance the survival of patients
with ARDS.
and macrophages have been extensively studied, the
involvement of lymphocytes (other immune cells)
recruited to the lungs has largely been ignored. Lympho-
cyte migration into the lungs was observed in ARDS dur-
ing the resolution of inflammation.2 Among the
lymphocytes, regulatory T-cells (Tregs) appear to pro-
mote the resolution of ARDS and accelerate tissue
repair.3,4
Regulatory T-cells, a distinct population of lympho-
cytes, play a critical role in maintaining immune home-
ostasis. Classically, Tregs have been associated with
various types of immune responses but, recently, they
were verified to affect diverse non-immunological con-
texts as well. For example, Tregs promote the regenera-
tion of acute and chronic muscle injury, improve healing
after myocardial infarction, and play a major role in
asthma and chronic obstructive pulmonary disease.5,6 At
Inappropriate accumulation of leucocytes and platelets,
dysregulated inflammation, and altered permeability of
Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; LPS,
lipopolysaccharide; Th, T helper; Tregs, regulatory T-cells
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162
151

W. Tan et al.
the sites of damage, Tregs exert a direct effect on their
progenitors, exhibit an indirect influence by regulating
macrophage activity7,8 and the balance of other T-cell
subpopulations, or prevent tissue damage through the
production of Amphiregulin by alarmin IL-18/IL-33,
which is distinct from immune responses suppression.4
Other specialized subpopulations of CD4+ T lympho-
cytes, including T helper (Th)1, Th2 and Th17 cells, have
been reported to play a significant role in the initiation of
immune responses by interacting with other cells9 and to
be involved in several inflammatory immune-mediated
disorders, mostly chronic inflammatory disorders. How-
ever, regarding bacteria-induced infectious acute inflam-
mation, the activities of these Th cells are ill defined. Th2
cells have been found to promote acute and chronic
inflammatory responses against various allergens and hel-
minths.10 In addition, a previous study confirmed that
IFN-ɣ+ Th1 cells are involved in the early stage of acute
lung injury (ALI)-induced inflammation.11 Th1 cells pro-
duce high levels of IFN-ɣ, are responsible for phagocyte
activation and play an important role in protecting
against intracellular pathogens. T helper-17 (Th17) cells
produce abundant inflammatory cytokines, including IL-
17a, IL-17f and IL-22, and are key mediators in host
defence, inflammatory disorders and autoimmune condi-
tions.12 Th1 and Th17 lymphocytes also appear to play a
significant role in neutrophilic airway inflammation. Acti-
vation of these immune cells is closely modulated by
counterregulatory circuits governed by Tregs, which
attenuate the overactive immune responses.13
Animals and methods
Animals
Adult male BALB/c mice at 6–8 weeks old were pur-
chased from the Animal Center of Peking Union Medical
College Hospital (Beijing, China). All animals were kept
in a specific-pathogen-free environment and maintained
on standard mouse chow at an environmental tempera-
ture of 22–24°, with 12-hr light and 12-hr dark cycles.
Male mice were randomly allocated into six groups as
follows: sham group; LPS-12-hr group (L12 h); LPS-1-
day group (L1); LPS-2-day group (L2); LPS-4-day group
(L4); and LPS-7-day group (L7). All mice were anaes-
thetized with intraperitoneal injection of 2% pentobarbi-
tal sodium (45 mg/kg body weight), then, the mice
received an intratracheal instillation of LPS (from Escheri-
chia coli serotype O55:B5; Sigma-Aldrich, St Louis, MO,
USA) at a dose of 3 mg/kg. Mice in the sham group
received only sterile saline (1ꢀ5 ml/kg).
This study was conducted in accordance with the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals and the Animal Management
Rules of the Chinese Ministry of Health. All experiments
were approved by the Animal Care Committee of Peking
Union Medical College.
Treg depletion
To deplete Tregs, mice were intraperitoneally injected
with 100 lg of anti-CD25 antibody (PC61; Biolegend,
San Diego, CA, USA) 10 days before LPS exposure, and
repeatedly treated every 7 days for continuous Treg
depletion. IgG was used as a control. Male mice were
randomly divided into 2-day-old and 4-day-old groups.
Every group was then further divided into four sub-
groups: Saline + IgG; Saline + anti-CD25; LPS + IgG;
and LPS + anti-CD25.
The resolution of pulmonary inflammation and the
process of tissue repair rely on a variety of immune and
non-immune cells and signalling molecules. Until now,
there has been little evidence of the direct effect of Tregs
on inflammation resolution and pulmonary repair in
ARDS. In addition, the role of Tregs in the pathophysiol-
ogy of ARDS has not been elucidated, and the reciprocal
relationship between Tregs and other subpopulations of
Th cells has remained elusive. Reviewing various factors
of ALI/ARDS, bacterial infection is the leading cause, and
lipopolysaccharide (LPS) as a cell wall component of
gram-negative bacteria is considerably important in the
pathogenesis of ALI/ARDS. Intratracheal instillation of
LPS is a well-accepted pathway to establish experimental
lung injury. Therefore, in this study, we used a mouse
model of LPS-induced ARDS to investigate the role of
Tregs in modulating the host response and tissue repair
in ARDS, and to comprehensively explore the mecha-
nism. We explicitly demonstrated that Tregs promote tis-
sue repair and the resolution of LPS-induced pulmonary
inflammation by promoting Th1 and Th17 cell responses.
In addition, we showed that in vivo depletion of Tregs by
injection of PC61 anti-CD25 antibody resulted in dimin-
ished Th1 and Th17 immune responses after LPS admin-
istration.
Bronchoalveolar lavage
The experimental procedure is shown in Fig. 2a. The
mice were killed, and bronchoalveolar lavage fluid (BALF)
was collected by lavage of the left lung. BALF was cen-
trifuged for 10 min at 300 g. The supernatant was
removed and stored at ꢁ80° for further detection. Total
BALF cells were counted using a haematocytometer.
Histopathological analysis
The middle lobe of the right lung was fixed in 4%
paraformaldehyde. The tissue was completely embedded
in paraffin, cut into small sections, stained with haema-
toxylin and eosin (HE), and measured by optical
152
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Tregs repair by modulating Th immunity
microscopy. Lung injury histopathological scores were
measured according to a previous study.14
Biosystems, Foster City, CA, USA). The relative expres-
sion levels of target genes were quantified using the DDCt
method and normalized to GAPDH genes (b-actin or
GAPDH) as described previously. The primer sequences
are shown in Table 1.
Isolation of lung and spleen cells
The right lung was removed, dissected into small sections
and incubated at 37° in RPMI 1640 medium containing
1 mg/ml collagenase I (Gibco/Invitrogen, Carlsbad, CA,
USA) and 50 lg/ml DNase (Sigma-Aldrich) for 1 hr.
Then, the tissues were passed through a 70-lm nylon cell
strainer (BD Falcon). The spleen was collected, ground
and mechanically dissociated in cold phosphate-buffered
saline (PBS). Samples were centrifuged at 300 g for 6 min
at 4°, washed and resuspended in PBS after lysis of red
blood cells.
Statistical analysis
Data are presented as the mean ꢂ SEM. All data were
analysed using one-way analysis of variance with a Bon-
ferroni post hoc test for multiple t-tests. A value of
P < 0ꢀ05 was considered statistically significant.
Results
Tregs accumulation is increased in LPS-induced
ARDS
Flow cytometry
The lung and spleen cells were stimulated with Leucocyte
Activation Cocktail (BD Pharmingen, San Jose, CA, USA)
for 6 hr when intracellular cytokines were detected. Cell
staining was performed with CD16/CD32 Fc, CD3, CD4,
CD25, CD31, CD326, CD45, F4/80 (Biolegend), Ly6C,
Ly6G, CD11b (eBioscience, San Diego, CA, USA). Cells
were fixed and permeabilized using a fixation/permeabi-
lization kit (eBioscience) or the BD Cytofix/Cytoperm
TM Fixation/Permeabilization Solution Kit (BD Pharmin-
gen) according to the manufacturer’s instructions. Then,
cells were stained for 30 min at 4° with IFN-ɣ, IL-17A,
IL-4 or Foxp3 (Biolegend), Ki-67 (eBioscience). Stained
cells were washed twice and resuspended in 4%
paraformaldehyde. Analysis of cell marker expression was
performed using Accuri C6 (BD, Franklin Lakes, NJ,
USA). Data were analysed with Flowjo software.
Regulatory T-cells represent approximately 5% of the
CD4+ T-cell compartment in uninjured lungs of BALB/c
mice. ARDS generated by intratracheal administration of
LPS in mice leads to the dynamic accumulation of a dis-
tinct population of Tregs within 1 week. The number of
pulmonary Tregs significantly increased on day 1 after
LPS administration and remained high on day 2, followed
by a decrease on day 4. After 7 days of recovery, pul-
monary Tregs were essentially normalized (Fig. 1a–c). In
addition, irrespective of pulmonary injury, the increase in
splenic Treg number showed similar dynamic variation
Table 1. Primer sequences applied in real-time PCR
IL-6
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
Forward 50-30
Reverse 50-30
CAACGATGATGCACTTGCAGA
CTCCAGGTAGCTATGGTACTCCAGA
ACTCCAGGCGGTGCCTATGT
GTGAGGGTCTGGGCCATAGAA
AAGCGTCATTGAATCACACCTG
TGACCTCAAACTTGGCAATACTC
GGGGCCAGTACAGCCGGGAA
CTGGCTGAAGGCAGTCCGCA
TCAACCAGCACCAGACAGAGA
TCCACCAAGACCACATCCAC
GGATGTAAGTCGAGGCCCAAG
ATTGCAAAGGTAGTGCCCGGTA
ACGGAGATGGATGTGCCAAAC
AGCACCTTGGAAGCCCTACAGA
ACGGCCCTGGTTCTCATCA
TNF-a
IFN-c
IL-10
Bead-based immunoassays
Secreted soluble protein in BALF was detected by bead-
based immunoassays using a Th panel kit (Biolegend)
according to the instruction. Samples were collected by
Accuri C6 (BD). Data were analysed with Biolegend
T-bet
LEGENDplexTM
software.
GATA3
Il-4
RNA extraction and real-time polymerase chain reaction
According to the manufacturer’s instructions, total RNA
was collected from lung homogenates using the Eastepꢀ
Super Total RNA Extraction Kit (Promega, Madison, WI,
USA). The RNA concentration and the A260/A280 ratio
were determined using a UV spectrophotometer. Total
RNA (1 lg) was reverse-transcribed to cDNA using
GoScript Reverse Transcriptase (Promega). Real-time
quantitative polymerase chain reaction (qPCR) was per-
formed using GoTaq qPCR mix (Promega) on the
Applied Biosystems 7500 Fast system (Applied
ROR-cT
IL-17a
TGF-b
Foxp3
GAPDH
CCAAATTGTATTGCAGATGTTCCAC
GCAAAAGTGAGCTCCAGAAGG
TCTTCATTGCGGTGGAGAGTC
TGTGGAACTCTACCAGAAATATAGC
GAAAGCCCTGTATTCCGTCTC
CACCTATGCCACCCTTATCCG
CATGCGAGTAAACCAATGGTAGA
AGGTCGGTGTGAACGGATTTG
GGGGTCGTTGATGGCAACA
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162
153

W. Tan et al.
(Fig. 1a,d,e). Tregs in the spleen reflected a parallel
change in the lung, and the disturbance of Tregs recov-
ered within 1 week in the lungs and spleen.
anti-CD25 antibody every 7 days (Fig. 2a). First, flow
cytometry was performed to confirm the Treg depletion
effects after anti-CD25 treatment (Fig. S1a–d), and the
numbers of CD4+ effector T-cells in the lungs and spleen
were not interfered (Fig. S1e–i). Then, the histopathologi-
cal changes of mouse lungs were observed after HE stain-
ing. No obvious abnormalities were observed in the saline
group and the saline + anti-CD25 group. When com-
pared with those in the LPS group, mice in the
LPS + anti-CD25 group did not demonstrate exacerbated
Impairment of the resolution of pulmonary injury in
Tregs-depleted mice
Next, we chose 2-day and 4-day time points to further
identify the role of Tregs in LPS-induced ARDS. A Treg-
depleted mouse model was established by injection of
(a)
Sham
L1
L2
L4
L7
105 3·68
2·35 3·85
10·4
6·01
14·6
5·21
8·38
8·67
4·68
104
103
102
101
Lung
80·9
1·64
4·80
14·5
84·9
2·36
1·53
14·3
85·8
3·75
0·88
9·88
93·9
100
0·12
7·98
76·7
1·81
2·70
15·4
105
4·36
104
103
102
101
Spleen
82·2
4·14
81·4
2·49
86·4
100
1·30
81·4
1·99
79·3
3·48
100 101 102 103 104 105 100 101 102 103 104 105 100 101 102 103 104 105 100 101 102 103 104 105 100 101 102 103 104 105
Gated on CD4+ T cells
CD25
Lung
Spleen
(b)
(d)
****
****
20
15
10
5
****
****
20
15
10
5
0
0
Sham L1
L2
L4
L7
Sham L1
*
L2
L4
L7
****
(c)
(e)
****
8
6
4
2
0
30
20
10
0
*
**
Sham L1
L2
L4
L7
Sham L1
L2
L4
L7
Figure 1. Regulatory T-cell (Treg) accumulation is increased in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS).
BALB/c mice were intratracheally injected with LPS. The experiment was repeated twice. (a) Cytofluorometric dot plots of Tregs within days after
injury. Numbers depict the fraction of CD4 + T-cells within the designated gate. (b) Summary data for the percentage and (c) number of Tregs
in the lungs depicted in (a). (d) Summary data for the percentage and (e) number of Tregs in the spleen depicted in (a). n = 8, Mean ꢂ SEM.
*P ≤ 0ꢀ05; **P ≤ 0ꢀ01; and ****P ≤ 0ꢀ0001.
154
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Tregs repair by modulating Th immunity
inflammatory histopathological changes and histopatho-
logical score on days 2 or 4 (Fig. 2b,c).
results showed a marked increase in Th17 cells in the
spleen of the LPS + anti-CD25 group compared with that
of the LPS group on day 2, the percentage of IL-17-pro-
ducing Th17 cells was markedly lower in the lungs of the
LPS + anti-CD25 group than in those of the LPS group,
especially on day 4 (Fig. 5a–c). The same results were also
observed in the spleen on day 4 (Fig. 5b,d). The concen-
trations of both IL-17a and IL-22 in the BALF were
clearly lower in the LPS + anti-CD25 group than in the
LPS group, and the decrease was especially profound on
day 4 (Fig. 5e,f). The level of IL17a was clearly lower in
anti-CD25 treatment after LPS administration on day 4
(Fig. 5g). A similar observation was noted regarding the
Total cell infiltration in BALF was detected after LPS
exposure by using haematocyte counter. Total cell counts
peaked on day 2 and decreased on day 4 after LPS
administration, Treg depletion attenuated the total cell
infiltration both on day 2 and day 4 after LPS administra-
tion, especially on day 2 (Fig. 2d). The cell types were
further differentiated via flowcytometry sorting of neu-
trophils, macrophages in lung suspension followed by
previous study15 (Fig. 2e), and T lymphocytes were also
detected. Treg depletion downregulated neutrophils and
upregulated macrophages on day 2 and day 4 after LPS
administration, without influencing
(Fig. 2f–h).
T
lymphocytes
expression of ROR-ct, a Th17 transcription factor
(Fig. 5h).
Finally, we examined the Th2 response and found
Tregs did not regulate Th2 immune responses during
LPS-induced pulmonary inflammation (Fig. S2).
Pulmonary epithelium is impaired and endothelial
proliferation is abrogated in Treg-depleted mice
Multicolour flow cytometry was used to identify specific
pulmonary epithelium and vascular endothelium in single
lung cell suspensions as follows: epithelium (CD45ꢁ
CD326+ CD31ꢁ); endothelium (CD45ꢁ CD326ꢁ CD31+).
In addition, the proliferation of epithelium or endothe-
lium was detected by marker of ki67. In our study, we
detected that the number of epithelial cells was decreased
on day 4 but not on day 2 in the LPS + anti-CD25 group
(Fig. 3a–c), and no significant difference in the number
of endothelial cells was observed in Treg-depleted mice
after LPS administration (Fig. 3d,e). After injection of
anti-CD25 antibody, although epithelial proliferation was
not affected when mice received an intratracheal instilla-
tion of LPS, endothelial proliferation was impaired on
day 4 (Fig. 3f,g). The schematic gating of ki67 + epithe-
lium or endothelium was presented by flow cytometry
(Fig. 3h).
Tregs might modulate Th responses via TGF-ß1
signalling
To further explore the mechanism of Tregs in the modu-
lation of Th responses, the representative proinflamma-
tory cytokines IL-6, TNF-a, TGF-b1 and IL-10 were
examined by real-time PCR and bead-based immunoas-
says. LPS administration led to increased expression and
secretion of both IL-6 and TNF-a in mouse lungs and
BALF on day 2. However, the expression and release of
IL-10 in the lungs were not statistically significant (data
not shown). The expression of IL-6/ TNF-a and release of
IL-6/ TNF-a into the BALF in the LPS + anti-CD25
group was markedly increased compared with that in the
LPS group on day 2 (Fig. 6a–d). However, the expression
and release of TGF-b1 were reduced in the LPS + anti-
CD25 group compared with the LPS group on day 4
(Fig. 6e,f).
Tregs promote Th1 and Th17 immune responses
during LPS-induced pulmonary inflammation
Discussion
To explore whether Tregs regulated LPS-induced pul-
monary inflammation by modulating immune responses,
the phenotype of CD4+ T lymphocytes in the lungs or
spleen and the levels of cytokines were evaluated. As
shown in Fig. 4, the percentage of IFN-c-producing Th1
cells was decreased in the lungs or spleens of anti-CD25-
treated mice on day 4 after LPS administration (Fig. 4a–
d). Anti-CD25 treatment reduced the concentration of
IFN-c in BALF on day 4 after LPS exposure but not on
day 2 (Fig. 4e). The variation of expression of IFN-c and
T-bet (Th1 transcription factor) determined by real-time
PCR was in accordance with the dynamic changes of
IFN-c in BALF (Fig. 4f,g).
In our study, Tregs promoted tissue repair and the reso-
lution of LPS-induced pulmonary inflammation by mod-
ulating the Th immune response. In vivo depletion of
Tregs by injection of PC61 anti-CD25 antibody resulted
in parallelly diminished Th1 and Th17 immune responses
after LPS administration.
Tregs were only recently identified, and participated in
the regulation of several T-cell-associated diseases, such as
inflammatory bowel disease and asthma.16,17 Foxp3 is a
key transcription factor and CD25 is a critical surface
marker regulating the development and maturation of
Tregs. Previous studies on Treg depletion used Foxp3DTR
mice and intraperitoneal administration of diphtheria
toxin1,18 or anti-CD25 neutralization. As CD25 mAb
(PC61 mAb) has been confirmed to deplete Foxp3+
The Th17 response was also studied in mice treated
with anti-CD25 (Fig. 5). Although the flow cytometry
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162
155

W. Tan et al.
(a)
anti-CD25 i.p anti-CD25 i.p
–3 day 0 day 2 day 4 day
–10 day
Sacrifice mice and harvest tissues
LPS+IgG
LPS I.T
(b)
Saline+IgG
Saline+anti-CD25
LPS+anti-CD25
2 day
4 day
(c)
(d)
200
150
100
50
0·6
0·4
0·2
0·0
***
Saline+IgG
Saline+anti-CD25
LPS+IgG
*
LPS+anti-CD25
0
2 day
4 day
2 day
4 day
(e)
Macropgages
Neutrophils
Ly6c
Ly6G
(f)
(g)
(h)
80
60
40
20
0
100
80
60
40
20
0
25
20
15
10
5
****
Saline+IgG
Saline+anti-CD25
LPS+IgG
**
**** ****
**** ****
**** ****
4 day
LPS+anti-CD25
0
2 day
4 day
2 day
4 day
2 day
Figure 2. Impairment of the resolution of pulmonary injury in regulatory T-cell (Treg)-depleted mice. (a) Schematic diagrams of the experimen-
tal design. Mice were intraperitoneally injected with 100 lg of anti-CD25 antibody or IgG 10 days before lipopolysaccharide (LPS) exposure and
repeatedly treated every 7 days for continuous Treg depletion. Mice were killed on 2 or 4 days after LPS administration. (b) Pulmonary
histopathological changes in mice as determined with haematoxylin and eosin (HE) staining. Representative images of the lungs (magnification
1009 and 2009). (c) Pulmonary histopathological score of (b). (d) Total cell counts in bronchoalveolar lavage fluid (BALF) were evaluated by a
haemocytometer. (e) Schematic sorting of CD11b+Ly6C+ Ly6G+F4/80- neutrophil and CD11b+Ly6C+Ly6G-F4/80 + macrophages in lung sus-
pension. (f) Percentage of neutrophils, (g) macrophages and (h) CD3 + T lymphocytes in lung suspension were determined by flow cytometry.
n = 6–8, Mean ꢂ SEM. *P ≤ 0ꢀ05; **P ≤ 0ꢀ01; ***P ≤ 0ꢀ001; and ****P ≤ 0ꢀ0001.
156
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Tregs repair by modulating Th immunity
(a)
Saline+IgG
Saline+anti-CD25
4·22
LPS+IgG
LPS+anti-CD25
6·69
38·5
28·7
7·38
36·8
6·66
29·8
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
(h)
10
10
10
10
2 day
14·6
1
40·2
6
37·0
30·1
6
18·0
1
37·8
20·7
1
42·8
6
2
3
4
5
2
3
4
5
2
3
4
5
6
1
2
3
4
5
10 10 10 10 10 10
10 10 10 10 10 10
10 10 10 10 10 10
10 10 10 10 10 10
CD45
5·93
39·2
4·83
35·6
12·4
16·9
6·97
17·2
5
5
4
3
2
1
5
5
4
3
2
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
4
3
2
1
4
3
2
1
10
10
10
10
4 day
14·6
1
40·2
6
19·2
1
40·4
6
33·8
1
36·9
6
Ki67
CD31
3·18
1
44·0
6
2
3
4
5
2
3
4
5
2
3
4
5
2
3
4
5
10 10 10 10 10 10
10 10 10 10 10 10
10 10 10 10 10 10
10 10 10 10 10 10
Gated on the CD45- cells
(d)
CD31
Saline+IgG
Saline+anti-CD25
LPS+IgG
(b)
20
(c)
60
50
40
30
20
20
LPS+anti-CD25
**
****
****
15
10
5
15
10
5
0
0
2 day
****
4 day
**
2 day
4 day
****
2 day
4 day
(e)
(f)
(g)
20
8
80
60
40
20
0
***
15
10
5
6
4
2
0
0
2 day
4 day
2 day
4 day
2 day
4 day
Figure 3. Pulmonary epithelium is impaired and endothelial proliferation is abrogated in regulatory T-cell (Treg)-depleted mice. (a) Cytofluoro-
metric dot plots of epithelial and endothelial cells within days after injury. Numbers depict the fraction of CD45- cells within the designated gate.
(b) Summary data for the percentage and (c) number of CD326 + epithelial cells in the lungs depicted in (a). (d) Summary data for the percent-
age and (e) number of CD31 + endothelial cells in the lungs depicted in (a). (f) Percentage of proliferating CD326 + cells and (g) percentage of
proliferating CD31 + cells in the lungs. (h) Schematic gating of ki67 + epithelium or endothelium. n = 6, Mean ꢂ SEM. *P ≤ 0ꢀ05; **P ≤ 0ꢀ01;
***P ≤ 0ꢀ001; and ****P ≤ 0ꢀ0001.
Tregs19–21 and PC61 has been shown to cause a signifi-
cant reduction in Foxp3+ Tregs by phagocytes via Ab-de-
pendent cellular cytotoxicity.22 In our study, we
employed the widely used and easily approachable strat-
egy of PC61 anti-CD25 mAb treatment to deplete
CD4+ FOXP3+ Tregs prior to LPS administration in mice.
Although PC61 does not lead to complete elimination of
Foxp3+ Tregs, the partial depletion of Tregs induced by
this method has been shown to adequately alter the
immunological balance for further exploration of the role
of Tregs.23 When considering CD25 is also expressed on
most activated T-cell subsets, we also explored the CD4+
effector T-cells in both lung and spleen. Our study
confirmed that 100 lg of anti-CD25 treatment con-
tributed to limit Foxp3+ Tregs generation without influ-
encing CD4+ effector T-cells.
Acute bacterial inflammation usually recruits neu-
trophils with a subsequent activation of macrophages,
which are critically involved in the first line of defence
against microbes. Neutrophils release inflammatory
cytokines, or proteinases, and cause the formation of
extracellular traps (NETs). Mitigated neutrophil infiltra-
tion induced delayed resolution of cutaneous wound
infection with advanced age in rat,24,25 and blocking
NETs formation reduced the capture of circulating bacte-
ria and aggravated the pathology that followed by a
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162
157

W. Tan et al.
Lung
Saline+anti-CD25
Spleen
Saline+anti-CD25
(b)
(a)
Saline+IgG
LPS+IgG
LPS+anti-CD25
Saline+IgG
LPS+IgG
LPS+anti-CD25
1·35
0·76
12·8
11·6
0·77
1·05
1·62
1·61
105
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
104
103
102
101
2 day
4 day
2 day
4 day
101 102 103 104 105
0·99
101 102 103 104 105
0·75
101 102 103 104 105
5·62
101 102 103 104 105
0·86
101 102 103 104 105
0·87
101 102 103 104 105
1·39
101 102 103 104 105
2·42
101 102 103 104 105
0·86
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
CD4
CD4
(c)
(d)
Spleen
Lung
****
3
2
1
0
15
****
****
Saline+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
LPS+anti-CD25
Saline+anti-CD25
LPS+IgG
LPS+anti-CD25
10
5
**
0
2 day
4 day
2 day
4 day
****
(e)
(f)
(g)
20
15
10
5
25
1500
**
****
*
***
Saline+IgG
Saline+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
LPS+anti-CD25
Saline+anti-CD25
LPS+IgG
Saline+anti-CD25
LPS+IgG
20
15
10
5
1000
500
0
LPS+anti-CD25
LPS+anti-CD25
0
0
2 day
4 day
2 day
4 day
2 day
4 day
Figure 4. Regulatory T-cells (Tregs) promote Th1 immune responses during lipopolysaccharide (LPS)-induced pulmonary inflammation. (a)
Cytofluorometric dot plots of IFN-ɣ+Th1 cells in the lungs. Numbers depict the fraction of Th1 cells within the designated gate. (b) Cytofluoro-
metric dot plots of IFN-ɣ+ Th1 cells in the spleen. Numbers depict the fraction of Th1 cells within the designated gate. (c) Summary data for the
percentage of Th1 cells in the lungs depicted in (a). (d) Summary data for the percentage of Th1 cells in the spleen depicted in (b). (e) Concen-
tration of IFN-ɣ in bronchoalveolar lavage fluid (BALF) was detected by bead-based Th immunoassays kit. (f) IFN-ɣ mRNA expression and (g)
T-bet mRNA expression in the lungs were assayed by real-time polymerase chain reaction (PCR). n = 6, Mean ꢂ SEM. *P ≤ 0ꢀ05; **P ≤ 0ꢀ01;
***P ≤ 0ꢀ001; and ****P ≤ 0ꢀ0001.
polymicrobial sepsis in vivo, resulting in increased dis-
semination to distant organs, even impaired vascular
remodelling.26 Treg depletion inhibited total cell infiltra-
tion, ameliorated neutrophil infiltration and promoted
macrophages recruitment, which might manifest impair-
ment of inflammation resolution and pulmonary repair.
The histopathological score of pulmonary section was
mainly aimed at exploring the inflammation response and
inflammation cells in the lungs. When considering pul-
monary repair, epithelial or endothelial cells in the lungs
were further detected by flow cytometry. In our study,
Treg depletion attenuated pulmonary repair by impairing
pulmonary CD326+ epithelial cells, which was consistent
with a previous study.1 The peak epithelial proliferation
was reported to occur on day 7 after I.T. LPS exposure,
this might be why we did not observe evident epithelial
proliferation when our experiment time was up to 4 days.
Although Treg depletion did not interfere with the
CD31+ endothelial cells, it damaged the proliferation of
endothelium in the lungs, which was in accordance with
reported elevation of pulmonary permeability when Tregs
were eliminated. However, we found there is a large per-
centage of CD45ꢁ cells that co-stain for both CD31 and
CD326, as from a previous study.27 This is maybe related
with activation of epithelium-to-endothelium transition
by enzyme digestion. Subgrouping of type 1 or 2 alveolar
epithelium is possible by adding additional marker to fur-
ther explore the co-stain cells.
Tregs influence the balance of Th1/Th2/Th17 during
various inflammatory diseases. Th1 immune responses are
considered to be crucial in orchestrating the host defence
against intracellular pathogens and in the pathogenesis of
many immune-mediated diseases.28,29 Th1 cytokines, such
as IFN-ɣ, TNF-a and IL-2, are released in response to
many innate stimuli, and can regulate both innate and
adaptive immune activities.30 T-bet is a critical regulator
of Th1 differentiation, and controls Th1 cell cytokine
production or migration by regulating the expression of
chemokines and their receptors.31 Tregs are generally
viewed as a suppressor of Th1 immune responses in
158
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162

Tregs repair by modulating Th immunity
Lung
Saline+anti-CD25
Spleen
(a)
(b)
Saline+IgG
LPS+IgG
LPS+anti-CD25
Saline+IgG
Saline+anti-CD25
LPS+IgG
LPS+anti-CD25
1·03
0·76
6·54
4·67
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
105
104
103
102
101
105
0·15
0·13
0·22
0·74
105
104
103
102
101
105
104
103
102
101
104
103
102
101
104
103
102
101
2 day
4 day
2 day
4 day
101 102 103 104 105
1·00
101 102 103 104 105
0·75
101 102 103 104 105
6·87
101 102 103 104 105
1·60
101 102 103 104 105
0·14
101 102 103 104 105
0·17
101 102 103 104 105
0·51
101 102 103 104 105
0·14
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
101
105
104
103
102
105
104
103
102
101
5 101
101 102 103 104 10
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
CD4
CD4
Lung
****
Spleen
(c)
(e)
(g)
(d)
15
10
5
****
***
***
1·0
0·8
0·6
0·4
0·2
0·0
Saline+IgG
Saline+anti-CD25
LPS+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
****
****
LPS+anti-CD25
LPS+anti-CD25
0
2 day
4 day
****
2 day
4 day
****
(f)
**
150
100
50
600
500
400
Saline+IgG
Saline+anti-CD25
LPS+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
*
LPS+anti-CD25
LPS+anti-CD25
300
300
200
100
0
0
2 day
*
4 day
***
2 day
4 day
**
(h)
600
400
200
0
15
10
5
*
Saline+IgG
Saline+IgG
***
Saline+anti-CD25
LPS+IgG
Saline+anti-CD25
LPS+IgG
*
LPS+anti-CD25
LPS+anti-CD25
0
2 day
4 day
2 day
4 day
Figure 5. Regulatory T-cells (Tregs) promote Th17 immune responses during lipopolysaccharide (LPS)-induced pulmonary inflammation. (a)
Cytofluorometric dot plots of IL17a+ Th17 cells in the lungs. Numbers depict the fraction of Th17 cells within the designated gate. (b) Cytofluo-
rometric dot plots of IL17a+ Th17 cells in spleen. Numbers depict the fraction of Th17 cells within the designated gate. (c) Summary data for
the percentage of Th17 cells in the lungs depicted in (a). (d) Summary data for the percentage of Th17 cells in the spleen depicted in (b). (e)
Concentration of IL-17a, (f) IL-22 in bronchoalveolar lavage fluid (BALF) was detected by bead-based Th immunoassays kit. (g) Il17a mRNA
expression and (h) ROR-ɣT mRNA expression were assayed by real-time polymerase chain reaction (PCR) in the lungs. n = 6, Mean ꢂ SEM.
*P ≤ 0ꢀ05; **P ≤ 0ꢀ01; ***P ≤ 0ꢀ001; and ****P ≤ 0ꢀ0001.
various inflammatory diseases.32,33 However, our finding
contradicts the result acquired by Liu et al.20 Differences
in the genetic background or the microbiome in the ani-
mal may lead to these divergent results; moreover, the
discrepant results may also be related to Th1 lymphocyte
plasticity.34,35 Although we observe a decrease in Th1
immune responses following LPS-induced pulmonary
damage in Treg-depleted mice, we could not exclude the
possibility that the kinetics of their elevation presence in
the lungs when the observed time points were prolonged.
Recent evidence has shown that Th17 immune
responses are crucial in protecting mucosal surfaces
against microbial pathogens, including bacteria, fungi and
viruses.36–38 Moreover, Tregs were shown to be tightly
correlated with the promotion of the Th17 immune
response in various models of inflammation. The levels of
Th17-related cytokines, such as IL-17a, IL-17f and IL-22,
were markedly increased in the LPS-induced mouse
model of ALI, inducing the pulmonary epithelium to pro-
duce chemokines that favour neutrophil infiltration,
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162
159

W. Tan et al.
(a)
(b)
15
10
5
1500
**
**
Saline+IgG
Saline+IgG
**
****
Saline+anti-CD25
LPS+IgG
Saline+anti-CD25
LPS+IgG
1000
500
0
LPS+anti-CD25
LPS+anti-CD25
0
2 day
4 day
2 day
****
4 day
(c)
(d)
***
****
500
250 000
200 000
150 000
100 000
50 000
0
Saline+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
Saline+anti-CD25
LPS+IgG
400
300
200
100
0
LPS+anti-CD25
LPS+anti-CD25
2 day
4 day
2 day
4 day
(e)
(f)
****
**
*
400
300
200
100
0
25
20
15
10
5
Saline+IgG
Saline+IgG
Saline+anti-CD25
LPS+IgG
Saline+anti-CD25
LPS+IgG
LPS+anti-CD25
LPS+anti-CD25
0
2 day
4 day
2 day
4 day
Figure 6. Regulatory T-cells (Tregs) might modulate Th responses via TGF-ß1 signalling. (a) Secretion of cytokine IL-6 in bronchoalveolar lavage
fluid (BALF). (b) IL-6 mRNA expression in the lungs. (c) Secretion of cytokine TNF-a in BALF. (d) TNF-a mRNA expression in the lungs. (e)
Secretion of cytokine TGF-ß1 in BALF. (f) TGF-ß1 mRNA expression in the lungs. n = 6, Mean ꢂ SEM. *P ≤ 0ꢀ05; **P ≤ 0ꢀ01; ***P ≤ 0ꢀ001;
and ****P ≤ 0ꢀ0001.
which is regarded as a proinflammatory mediator and
contributor to airway remodelling.16,17 Blockade of IL-17
may facilitate pulmonary fibrosis in asthma.39 However,
the relevance of Tregs in the induction of Th17 responses
against bacterial infection remains elusive. In a murine
model of Chlamydia muridarum-induced genital tract
infection, Tregs were determined to serve as a prominent
inducer of and direct contributor to IL-17/Th17
responses.40 Tregs were also shown to stimulate the
Th17-mediated inflammatory response in a CTLA4-de-
pendent manner in colon inflammation.41 During oral
Candida albicans infection in mice, Tregs were shown to
promote acute Th17 cell responses to suppress mucosal
fungus infections and maintain immune homeostasis.42 In
accordance with those findings, we confirmed that Tregs
could promote the Th17 immune response in LPS-in-
duced ARDS.
allergies and atopic illnesses. Th2 cells release various
pathophysiological cytokines (IL-4, IL-5, IL-13) and stim-
ulate type 2 immunity, such as in human asthma, as well
as in an animal model of allergic airway inflamma-
tion.43,44 However, type 2 immunity appeared to be lack-
ing in the bacterial infection mouse model. Consistent
with our observations, Treg depletion in bacterial infec-
tion has been shown to have a limited impact on Th2
responses.
As CD4+ Th subsets possess the characteristics of plas-
ticity, Foxp3+ Tregs have been shown to convert into Th1
cells or Th17 cells under certain conditions.45 Moreover,
Th17 cells highly expressing aryl hydrocarbon receptors
can convert into Tregs during the resolution of inflam-
mation in the presence of TGF-b.46 Th1 cells can also
convert into Th17 cells under inflammatory conditions in
the intestine mediated by TGF-ß-induced Runx1 expres-
sion.47 Therefore, Treg depletion might impair the recip-
rocal conversion among Treg, Th1 and Th17 cells in LPS-
Th2 cells are recognized for their role in host defence
against multicellular parasites, and are involved in
160
ª 2019 John Wiley & Sons Ltd, Immunology, 157, 151–162

Tregs repair by modulating Th immunity
induced pulmonary inflammation and lead to reduced
Th1 and Th17 subsets.
Tregs have been ascribed beneficial or detrimental roles
during infection depending on the nature of the infec-
tious agent or whether the infection is acute or chronic.
Taken together, the data show that Tregs promote tissue
repair and the resolution of LPS-induced pulmonary
inflammation by promoting Th1 and Th17 responses.
Moreover, our study provided vital supplemental evi-
dence for immune cell involvement and revealed the Th
response profile in LPS-induced pulmonary inflamma-
tion. In addition, our study showed the modulation of
the Th response by Tregs.
Importantly, Treg depletion after LPS administration
had an effect on the Th1 and Th17 immune responses on
day 4 in LPS-induced ARDS. Therefore, we concluded
that the time frame in which Tregs promote Th1 and
Th17 cell development and their function is probably not
short.
To investigate the mechanism by which Tregs regulate
the Th subpopulation, we detected the proinflammatory
cytokines IL-6, TNF-a, IL-10 and TGF-ß. IL-6 and TNF-a
are two significant proinflammatory factors used to evalu-
ate inflammation. The elevated level of IL-6 and TNF-a
confirmed that Treg depletion aggravated pulmonary
inflammation after LPS administration. The elevation was
detected on day 2, which is inconsistent with the time
frame of changes in Th1 and Th17. IL-10 and TGF-ß1
are two crucial factors secreted by Tregs and play vital
roles in promoting the differentiation of Th1 and Th17
cells in the proinflammatory cytokine-enriched microen-
vironment after infection. TGF-ß1 was highly expressed
by activated T-cells, more importantly implicated in
Treg-suppressive activity and Foxp3 expression,3 facili-
tated the differentiation of Th17 cells and controlling
inflammatory diseases,48,49 and enhanced effector Th1 cell
activation via indirect pathway.50 Although there was no
significant difference in the level of IL-10 in BALF and
the lungs, we found that the expression and release of
TGF-b1 were decreased in Treg-depleted mice 4 days
after LPS administration, paralleling the time at which
Th1 and Th17 variations are observed. Therefore, we pre-
dicted that Tregs might likely modulate Th1 and Th17
differentiation in a TGF-b1-dependent manner in a
mouse model of LPS-induced ARDS. As we lack the fur-
ther verified evidence, the TGF-ß1-related mechanism
involved in both cell and animal experiments would be
performed in our further study.
Acknowledgements
Wen Tan performed the experiments and wrote the
paper, Chaoji Zhang and Jianzhou Liu analysed the data,
and Qi Miao designed the study. This work was sup-
ported by a grant from the CAMS initiative for innova-
tion medicine (NO. CAMS-I2M-1-003).
Disclosures
The authors declare that there are no financial or com-
mercial conflicts of interest.
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Additional Supporting Information may be found in the
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Figure S2. Tregs do not regulate Th2 immune
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