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.