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1
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China.
2
Department of Anatomy, Zhejiang University School of Medicine, Hangzhou, 310058, China.
3
Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, Guangdong, 510632, China.
4
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China. [email protected].
5
International Institutes of Medicine, Zhejiang University School of Medicine, Yiwu, 322000, China. [email protected].
6
Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, 310058, China. [email protected].
7
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China. [email protected].
8
State Key Lab of Respiratory Disease, Guangzhou, 510120, China. [email protected].
1
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China.
2
Department of Anatomy, Zhejiang University School of Medicine, Hangzhou, 310058, China.
3
Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, Guangdong, 510632, China.
4
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China. [email protected].
5
International Institutes of Medicine, Zhejiang University School of Medicine, Yiwu, 322000, China. [email protected].
6
Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, 310058, China. [email protected].
7
Key Laboratory of Respiratory Disease of Zhejiang Province, Department of Respiratory and Critical Care Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, China. [email protected].
8
State Key Lab of Respiratory Disease, Guangzhou, 510120, China. [email protected].
Eosinophils are terminally differentiated cells derived from hematopoietic stem cells (HSCs) in the bone marrow. Several studies have confirmed the effective roles of eosinophils in asthmatic airway pathogenesis. However, their regulatory functions have not been well elucidated. Here, increased C-C chemokine ligand 6 (CCL6) in asthmatic mice and the human orthologs CCL15 and CCL23 that are highly expressed in asthma patients are described, which are mainly derived from eosinophils. Using Ccl6 knockout mice, further studies revealed CCL6-dependent allergic airway inflammation and committed eosinophilia in the bone marrow following ovalbumin (OVA) challenge and identified a CCL6-CCR1 regulatory axis in hematopoietic stem cells (HSCs). Eosinophil differentiation and airway inflammation were remarkably decreased by the specific CCR1 antagonist BX471. Thus, the study identifies that the CCL6-CCR1 axis is involved in the crosstalk between eosinophils and HSCs during the development of allergic airway inflammation, which also reveals a potential therapeutic strategy for targeting G protein-coupled receptors (GPCRs) for future clinical treatment of asthma.
Increased expressions of hCCL23 and hCCL15 in asthma patients.
a
Relative mRNA expressions of
hCCL23
and
hCCL15
in total white blood cells (WBCs) from asthma patients (
n
=16) versus healthy control subjects (
n
=16).
b
hCCL23 concentration measured by ELISA in the plasma of asthma patients (
n
=31) compared with healthy control (
n
=30). Data in
a
and
b
are presented as median (centerline), and the whiskers indicate no more than 1.5 times the interquartile range (the difference between 25th and 75th percentiles, shown in the box).
c
Correlation of hCCL23 protein levels with the number of eosinophils in human peripheral blood (
n
=61, linear regression and Spearman rank correlation).
d
Schematic illustration of peripheral blood WBCs isolating procedure from asthma patients.
e
Representative images of total WBCs and isolated subsets slides of Wright-Giemsa staining and immunofluorescence staining for EPX (green), hCCL23 (red), hCCL15 (red), and DAPI (blue). Scale bar, 40μm. Insets show high-power images in the upper right corner of each overlay. Scale bar, 10μm. **
P
<0.01 by unpaired
t
-test
Increased expressions of eosinophil-derived mCCL6 in allergen-challenged mice.
a
Sequence alignment of the mCCL6 with the hCCL23 and hCCL15.
Shaded letters
indicate residues at each aligned position that are identical to the hCCL23 and hCCL15 specificity.
Dashes
indicate gaps that were inserted to optimize the alignment.
b
Schematic timeline and subsequent sample processing of allergic asthma mouse models.
c-e
Expressions of mCCL6 in BALF supernatant (
c
), lung tissue (
d
), and serum (
e
) measured by ELISA from NS or OVA-challenged mice. Each point represents an individual mouse; data from 4–5 mice per group are plotted as mean ± SEM.
f
Correlation of mCCL6 protein levels with the number of eosinophils in murine BALF (
n
= 17, some of the points are overlapped).
g
Representative histograms of mCCL6 expression in monocytes, eosinophils, and neutrophils from peripheral blood of NS mice. Cells were gated in Fig. S2a.
h
Representative lung immunofluorescence of eosinophils (tdTomato
+
, red) in OVA-challenged eoCre/R26-tdTomato mice staining with mCCL6 antibody (green) and DAPI (blue). Scale bar, 40 μm, 20 μm.
i
Representative histograms of mCCL6 expression in BALF eosinophils from OVA-challenged mice compared with NS mice.
j
Representative cytospin slides of bone marrow exhibited eosinophils (tdTomato
+
, red) in NS or OVA-challenged eoCre/R26-tdTomato mice staining with mCCL6 antibody (green) and DAPI (blue). Scale bar, 40 μm.
k
Quantitation of mCCL6
+
percentage among tdTomato
+
cells in
j
(
n
= 6 per group, 3 images per mouse). **
P
< 0.01; ***
P
< 0.001 by unpaired
t
-test
CCL6 deficiency alleviates OVA-induced eosinophilic airway inflammation.
a
Schematic map of established
Ccl6
−/−
mice by using the CRISPR/Cas9 system.
b
Representative blots of mCCL6 and β-actin (loading control) assessed by Western blot of protein extracts of eosinophils sorted from WT and
Ccl6
−/−
mice.
c
Differential counts on Wright-Giemsa stained BALF cells in WT and
Ccl6
−/−
mice. OVA-induced asthma model was presented in Fig. 1a. Combined data shown as mean ± SEM are presented for 9–10 mice per group from two independent experiments. Representative photomicrographs of lung sections with H&E staining (
d
), EPX staining (
f
), and those with PAS staining (
h
) at 24 h after the last OVA challenge. Scale bar, 100 μm. Histological inflammatory scores (
e
) and PAS scores (
i
) were analyzed from
d
and
h
.
g
The quantitative percentages of EPX
+
cells in total nucleated cells analyzed from
f
(
n
= 4–5 mice per group, 4 images per mouse).
j
Relative mRNA levels of
Il-13
and
Il-25
in lung tissues were determined by quantitative RT-PCR at 24 h after the last NS or OVA challenge.
k
The concentration of IL-4 and IL-33 in lung tissue determined by ELISA. Data are mean ± SEM for 4–5 mice per group, 5–7 images per mouse. *
P
< 0.05; **
P
< 0.01; ***
P
< 0.001; ****,
P
< 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test
CCL6 deficiency abolishes the impairment of HSC homeostasis in allergen-induced airway inflammation.
a
Murine eosinophil differentiation hierarchy.
b-c
Quantitative number of eosinophils in peripheral blood (
b
) and bone marrow (
c
) of NS or OVA-challenged WT and
Ccl6
-/-
mice.
d-e
Representative flow cytometric dot plots (
d
) and quantitative number (
e
) of LSK.
f-g
Representative flow cytometric dot plots (
f
) and quantitative number (
g
) of CMP, GMP, MEP.
h
,
i
Representative flow cytometric dot plots (
h
) and quantitative number (
i
) of EoP. Data are statistically calculated as mean ± SEM for 4–6 mice in each group from three independent experiments. *
P
< 0.05; **
P
< 0.01; ***
P
< 0.001 by one-way ANOVA with Sidak’s multiple comparisons test
mCCL6 directly activates CCR1 and downstream signaling.
a
Schematic illustration of GloSensor assay in CCR1. Forskolin artificially elevated cAMP levels by activating adenylyl cyclase (AC) whereas CCR1 agonist inhibits AC activity.
b
Dose-response curves of the intracellular cAMP level measured by GloSensor assay. GloSensor-HEK293T cells transfected with vehicle or mCCR1 were treated with forskolin (1 μM) and indicated concentrations of mCCL6. The reduction of cAMP was recorded after 30 min. Data represent mean ± SEM of three technical replicates (error bars smaller than symbols are not shown). Median effective concentration (EC50) was calculated by nonlinear regression (three parameters).
c
,
d
Representative blots of p-ERK1/2, ERK1/2, p-p38, p38 and GAPDH (loading control) assessed by Western blot of protein extracts from mCCR1-293T (
c
) or vehicle-293T (
d
) cells cultured with 200 ng/ml mCCL6
Inhibition of CCR1 alleviates OVA-induced lung eosinophilic inflammation.
a
Schematic of BMDE processing and culture timeline representing the treatment of BX471.
b
Number of eosinophils characterized as SiglecF
+
F4/80
+
cells in WT BMDEs treated with BX471 as indicate in
a
(
n
= 4 mice per independent cultures).
c
Schematic timeline representing the treatment of BX471 in allergic asthma models.
d
Differential cell counts of cytospin preps represented as the number of BALF cells.
e
Representative images of lung tissues immunostained with EPX antibody after therapeutic treatment with BX471. Scale bar, 100 μm.
f
The quantitative percentages of EPX
+
cells compared with total nucleated cells analyzed from
e
(
n
= 4–5 mice per group, 4 images per mouse).
g
,
h
Relative mRNA levels of
Il-13
and
Il-25
(
g
) and protein levels of IL-4 and IL-33 (
h
) in lung tissues determined by quantitative RT-PCR and ELISA. Data are mean ± SEM for 4–5 mice in each group from two independent experiments in
d
,
f
–
h
. n.s., not significant; *
P
< 0.05; **
P
< 0.01 ***
P
< 0.001; ****
P
< 0.0001 by two-way ANOVA in
b
, one-way ANOVA with Tukey’s
post hoc
test in
d
,
f
–
h
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