Materials and methods
Mice
6‐week‐old female C57BL/6 mice were obtained from Silaike Experimental
Animal Limited Liability Company (Shanghai, China). All animals were
treated in accordance with the National Institutes of Health Guidelines
and Regulations.
Isolation and characterization of BMDCs from mice
BMDCs were differentiated from BM cells obtained from femurs and tibiae
of C57BL/6 mice. Briefly, BM cells were seeded in RPMI-1640 supplemented
with 1% antibiotics/antimycotics and 10% heat-inactivated fetal calf
serum (FCS) containing 20 ng/ml granulocyte-macrophage
colony-stimulating factor (GM-CSF). The total culture period was 9 days.
On day 3, fresh medium was added at the same volume as the preexisting
medium. On day 6, half of the culture medium was replaced with fresh
medium. On day 8, nonadherent DCs were resuspended in complete medium
supplemented with GM-CSF alone (to generate immature DCs; DCia) or with
50 ng/mL IL-10 or 10 ng/mL LPS (to generate tolerogenic cells; DC10 and
DClps) for 24 h. On day 9, cells from each culture were pulsed for 2 h
at 37℃ with 1 μM OVA (Sigma-Aldrich, Grade V; St Louis, MO, USA) and
then the cells were harvested. Meanwhile, noninduced BM cells were
collected and cultured under the same conditions except that there was
no GM-CSF in the culture medium for 9 days.
Harvested BMDCs and BM cells were characterized by evaluating the
expression of CD11c, CD80, and MHCII. Cells were washed and stained at
4℃ for 30 min with APC-Cy7 conjugated anti-CD11c, APC-conjugated
anti-CD80, and FITC-conjugated anti-MHCII (eBioscience, Inc., San Diego,
CA, USA) and were subsequently analyzed by flow cytometry (FACS Canto
II; BD Biosciences, San Jose, CA, USA) to determine the positive rate
for the expression of labeled antigens. All data were analyzed with Flow
Jo software.
Establishment of OVA-induced asthmatic mice and adoptive transfer of
diverse BMDCs into asthmatic mice
To establish an asthma phenotype, 6-week-old female C57BL/6 mice were
intraperitoneally
sensitized with PBS containing 100 µg OVA in 2 mg alum or PBS/alum as a
control on days 0 and 7. Animals were then intranasally challenged with
100 μg OVA in PBS or PBS as a control under anaesthetic on days 14-18.
For the treatment models, 1×106 cells (DCia, DC10 or
DClps prepared as described above) were intraperitoneally transferred on
days 14-16.
At 24 h after the last challenge, mice were anesthetized, and
bronchoalveolar lavage (BAL) was performed with cold PBS. Subsequently,
lungs were obtained for histology and fixed with 4% paraformaldehyde in
PBS. BALF was centrifuged (1200 rpm for 10 min, at 4°C), and cells were
obtained to assess the total cell number and differential cell counts.
All samples were independently processed and assayed.
Isolation of memory CD4+ T cells from OVA-induced asthmatic mice
We established another asthma
phenotype
in mice to obtain pulmonary memory CD4+ T cells. The OVA sensitization
and challenge protocol was similar to the previous protocol, and BMDCs
were adoptively transferred to OVA-sensitized mice for 3 consecutive
days. However, the mice were not sacrificed on day 19. Mice were
rechallenged with OVA for 5 consecutive days (day 35-39) and sacrificed
on day 40 for the detection of memory T cells. We generated single-cell
suspensions of lung parenchymal cells by enzymatically digesting
tissues, and identified pulmonary memory CD4+ T cells with flow
cytometry. The phenotypic analysis of effector memory CD4+ T cells and
central memory CD4+ T cells were performed by evaluating the expression
of CD4, CD44, CD62L, and CCR7. A total of 1×106 cells
were washed and subsequently stained with FITC-conjugated anti-CD4,
PE-conjugated anti-CD44, APC-conjugated anti-CD62L and
PerCP-Cy5.5-conjugated anti-CCR7 (eBioscience). All data were analyzed
with Flow Jo software.
Mixed lymphocyte reaction (MLR) of memory CD4+ T cells and BMDCs
Pulmonary memory CD4+ T cells and BMDCs were prepared as described
above. BMDCs were pretreated with culture medium containing 200 μg/mL
mitomycin C for 20 min at 37°C (Kyowa Hakko Kogyo, Tokyo, Japan).
Pulmonary memory CD4+ T cells were labeled with carboxyfluorescein
diacetate succinimidyl ester (CFSE, Invitrogen Ltd., UK) prior to
culture to evaluate T cell proliferation. Mitomycin C-treated DCs
(l×105/well) and isolated memory CD4+ T cells
(4×l05/well) were cocultured in 96-well plates at a
ratio of 1:4 at 37°C, 5% CO2 atmosphere for 1, 3 or 5
days respectively. The coculture systems were divided into five groups:
the control group, the OVA group, the OVA+DCia group, the OVA+DC10 group
and the OVA+DClps group, and BMDCs were generated as previously
described. After MLR, the proliferation activity of memory CD4+ T cells
was evaluated with CFSE on days 1, 3, and 5. On the last day, cells were
stimulated with phorbol myristate acetate (PMA; 100 ng/ml, Sigma) and
Inomycin (5 µmol/L, Sigma) overnight, and then cell supernatants were
collected to assess IL-4 and IFN-γ secretion by ELISA.
Pulmonary Tregs of OVA-induced asthmatic mice after the adoptive
transfer of DClps
As noted, lung tissues of asthmatic mice that were sacrificed on day 19
were immediately collected to determine the number of Tregs in the lung.
Single-cell suspensions of lung parenchymal cells were generated by the
enzymatic digestion of tissues, and Tregs were characterized as
CD4+CD25+Foxp3+ cells by flow cytometry. A total of
1x106 cells were washed and stained with
FITC-conjugated anti-CD4, PE-conjugated anti-CD25 and APC-conjugated
anti-Foxp3 (eBioscience) antidodies, and subsequently analyzed by flow
cytometry. All data were analyzed with Flow Jo software.
BMDC tracking in the lung
We labeled transferred DCs with the fluorescent dye PKH26
(Sigma‐Aldrich) according to the instructions. Briefly, we used 1 mL
dilution buffer to suspend 2×106 DCs and then mixed
the cell suspension with the same amount of labeling solution containing
4×10-6 M PKH26 dye in dilution buffer. After 4 min of
incubation at room temperature, we added 2 mL fetal bovine serum (FBS)
to terminate the reaction and washed the cells with a control buffer.
Finally, 5 × 106 DCs labeled with PKH26 dye were mixed
in 1 mL PLA‐CMC solution for the subsequent adoptive transfer.
PKH26-labeled DCs were intraperitoneally injected into asthmatic mice
(1×106 cells/mouse), as described above. After 1, 3
and 7 days, we obtained lung tissues from each animal and acquired
single-cell suspensions by the enzymatic dispersal of the lungs.
Delivered DCs in the lung were assessed via the expression of MHCII and
PKH26 as determined by flow cytometry. All data were analyzed with Flow
Jo software.
Histopathologic analysis of the lung
The left lung was removed from each mouse and infused with 4%
paraformaldehyde after BAL, embedded in paraffin, sectioned at 5 um, and
stained with hematoxylin and eosin (H&E) according to standard
procedures for evaluating histopathological changes in bronchial and
lung tissues. Right lung specimens were collected and frozen at -80°C
for protein analysis by western blotting (WB).
Peribronchial and perivascular inflammation were estimated with a
subjective scale of 0–3 based on a scoring system: a score of 0
indicated that there was no detectable inflammation; 1 indicated that
most bronchi or vessels were surrounded by a thin layer (one to five
cells thick) of inflammatory cells; 2 indicated that most bronchi were
surrounded by a thin layer (1–5 cells) of inflammatory cells; 3
indicated that most bronchi or vessels were surrounded by a thick layer
(>5 cells) of inflammatory cells18. The
average peribronchial and perivascular inflammation scores reflected the
total lung inflammation of treated animals. The score is presented as a
mean value of each animal.
Cytokine release capacity of BMDCs
BMDCs (DCia, DC10, and DClps) were harvested after 9 days of culture, as
described above. Cells from each culture were pulsed for 2 h at 37℃ with
1 μM OVA. The cell culture supernatant was collected and stored at -80℃
for subsequent cytokine detection of IL-10, TGF-β, MCP-3 and IFN-γ by
ELISA (R&D Systems).
WB analysis
Expression levels of STAT1, 4, and 6 and their phosphorylated protein
levels in lung digests were assessed by WB. Total protein from mouse
right lung tissue was extracted by using RIPA Protein Extracted Reagent
(Thermo). 40 μg lysate samples were fractionated by 10% sodium dodecyl
sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF)
membranes (Roche, USA). After incubation with blocking buffer containing
5% skim milk in TBST (12.5 mM Tris-HCl pH 7.5, 68.5 mM NaCl, 0.1%
Tween 20) for 1 h, the membrane was then incubated with primary
antibodies overnight, including rabbit anti-STAT1, and rabbit
anti-phosphorylated STAT1, rabbit anti-STAT4, rabbit anti-phosphorylated
STAT4, rabbit anti-STAT6, rabbit anti-phosphorylated STAT6 antibodies.
The anti-mouse GAPDH antibody was used as a loading control. The
membrane was washed with TBST and incubated with
horseradish-peroxidase-conjugated secondary antibody (Jackson
ImmunoResearch) and then developed with and electrochemiluminescence
(ECL) substrate solution (Millipore). The membrane was incubated with
rabbit anti-murine GAPDH for assessment of internal protein loading
control after incubation with stripping buffer for 15 min at 25℃. The
density of each band was quantified using image analysis software
(ImageJ).
Statistical analysis
All data are representative of at least three independent experiments.
Each error bar represents the standard deviation (SD). Multigroup
comparisons were assessed by either one-way ANOVA with Tukey’s post hoc
test or by Wilcoxon signed rank tests (FACS analyses) with a commercial
software program (GraphPad Prism 8.0, San Diego, CA). P < 0.05
was considered statistically significant.