Induced differentiation of THP-1 macrophages
We cultivated human acute monocytic leukaemia mononuclear THP-1 cells with the cell density adjusted to 1.0×106/ml. TPH-1 cells were induced to differentiate into adherent THP-1 macrophages by phorbol-12-myristate-13-acetate (PMA, 100 ng/ml) stimulation for 24 h, mimicking human AMs.
Plasmid construction and cell transfection
siRNA-mediated CD23/PU.1 interference and pRK5-HA (Promega, US) were used to silence and overexpress CD23/PU.1 expression, respectively. The siRNA sequences against CD23 were sense 5’-GGAGGAACUUCGAGCUGAACA-3’ and antisense 5’- UUCAGCUCGAAGUUCCUCCAG-3’. The siRNA sequences against PU.1 were sense 5’-CAAGUAAAGUUAUUCUCAAUC-3’ and antisense 5’-UUGAGAAUAACUUUACUUGUU-3’. THP-1-derived macrophages were transiently transfected with CD23/PU.1 siRNA and pRK5-CD23 with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, US) according to the manufacturer’s instructions controlled by a negative control.
Observation of phagocytic ability of the macrophages
THP-1 macrophages with CD23 silencing and overexpression were incubated with FITC-labelled AFA1 (AF strains used in this study, referred to as AF) conidial suspensions (multiplicity of infection (MOI) = 1) for 4 hours. The nuclei and cell membranes of the macrophages were stained with 4',6-diamidino-2-phenylindole (DAPI) (Solarbio, C0065) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) (Absin, abs47014947) fluorescent probes. A laser scanning confocal microscope (Lecia SP8, Germany) was used to observe the phagocytic ability of THP-1 macrophages to AF.
Establish of the AF infection model
THP-1 macrophages were incubated with AF conidial suspensions (MOI = 1). Macrophages were harvested at 0 h, 8 h, 16 h and 24 h after AF conidial stimulation. Quantitative real-time polymerase chain reaction (qRT–PCR), Western blotting analysis and enzyme-linked immunosorbent assay (ELISA) were performed to quantify CD23 and PU.1 expression and inflammatory factor levels.
qRT–PCR
Total RNA was extracted from THP-1-derived macrophages with TRIzol reagent. The mRNA was reverse transcribed to cDNA and subjected to qRT–PCR with the PrimeScript® RT Master Mix Perfect Real Time kit (TAKARA Bio Inc., Kusatsu, Japan) and SYBR Green Master Mix (Applied Biosystems, Foster City, CA, US). The reaction was conducted on a qPCR instrument (Applied Biosystems 7900HT Real-Time System, US). The relative gene expression levels were analysed by the 2-ΔΔCt method and normalized to actin. The primer sequence information was forward primer 5’-GCACCTTCCAGTTCTCGTCCAAGC-3’ and reverse primer 5’-CGCCGCTGAACTGGTAGGTGAGCT-3’ for PU.1 and forward primer 5’-ACTGCGTGATGATGCGGGGCTCC-3’ and reverse primer 5’-GTCAGGGTCTGGTCTTGAATCAG-3’ for CD23.
Western blotting analysis
The collected macrophages were lysed with RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with protease inhibitor cocktail (Roche, Pleasanton, CA, US). The lysis products were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels for electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, blocked in 5% milk, and incubated with primary antibodies (1:1000, rabbit anti-CD23, rabbit anti-PU.1) (Abcam Inc., Cambridge, MA, US) and secondary antibodies (1:5000, goat anti-rabbit IgG) (Abcam Inc., Cambridge, MA, US). Autoradiograms were quantified by densitometry with GAPDH as a control.
ELISA
Intracellular inflammatory factor levels were detected using an ELISA kit (Solarbio, Beijing) according to the manufacturer's instructions.
CD23-expressing adenovirus
The recombinant adenovirus plasmid pAD-CD23-pIRES2-ERFP was obtained and transfected into human embryonic kidney 293 (HEK293) cells after linearization by Pac I. HEK293 cells amplified the recombinant adenovirus AD-CD23-ERFP (referred to as AD-CD23) in large quantities. The recombinant adenovirus AD-ERFP served as a control.
Animal experiment
To investigate the effect of CD23 in mouse lungs, we anaesthetized BALB/c mice with pentobarbital (70 mg/kg) and transfected 30 μl CD23-expressing adenovirus AD-CD23 (~3×108 PFU) into the mice via trachea controlled by adenovirus expressing ERFP (Ad-ERFP). CD23 mRNA expression in lung tissues of each group was detected by qRT–PCR.
CD23-upregulated mice were obtained following the above method, and wild-type mice were injected intraperitoneally with cyclophosphamide (200 mg/kg) 1 d after adenovirus AD-CD23 administration. The immunodeficiency mouse model was established after 4 d of continuous cyclophosphamide administration. Immunodeficient mice were intratracheally injected with AF conidia (5×106 PFU) controlled by normal saline treatment. The mice were sacrificed with pentobarbital (70 mg/kg) 7 d after AF infection. The diseased lung tissue in mice was separated, and haematoxylin and eosin (HE) staining and immunochemistry analysis were performed to detect the pathological changes in the mouse lung tissues.
Immunohistochemistry
Paraffin-embedded blocks were cut into 4-μm thick sections. The dewaxed and hydrated tissue sections were incubated with anti-PU.1 (Abcam, ab88082) or anti-CD23 (Abcam, ab254162) antibody for 2 h at room temperature and subsequently incubated with a goat-anti-rabbit antibody for 40 min. The degree of staining was determined by development with diaminobenzidine (DAB) chromogen (Bio-Rad, Inc., CA, USA) and detection using a microscope (Olympus, Japan).
Luciferase assay
pRK5-PU.1 cells overexpressing PU.1 and pGL3-CD23 with the LUC reporter were constructed and transfected into HEK293T cells with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, US). Twenty-four hours after transfection, the HEK293T cells were incubated with AF conidia for 12 h. The LUC signals were analysed by a dual luciferase reporter gene detection kit (Promega, US) and FlowJo V10 software (Ashland, USA).
ChIP
THP-1 macrophages and AF conidia were co-incubated (MOI = 1) for 8 h. Then, formaldehyde (1%) was applied to crosslink the proteins and chromatin for 10 minutes at room temperature. After that, the macrophages were lysed with an Ultrasonic Breaker (Bioruptor, Belgium). Immunoprecipitation was performed with an IP-level PU.1 antibody (Abcam, US) and an EZ ChIP kit (Millipore, Germany). qPCR was performed to detect the binding activity of PU.1 to the promoter of CD23 after the immune precipitate was washed.
EMSA
According to the ChIP results, biotin-labelled probes for different sites were designed. The nonbiotin-labelled probes were applied as competitive controls with 25-fold, 50-fold and 100-fold competition concentrations. Total proteins were extracted with a cytoplasm-nucleus protein extraction kit (KeyGEN BioTECH, Nanjing, China) following the instructions. The nucleoprotein and nucleic acid probes were combined and reacted. After electrophoresis, membrane transformation and ultraviolet crosslinking, chemiluminescence was used to detect the binding activity of PU.1 to CD23.
Statistical analysis
SPSS 20.0 and SigmaPlot 12.0 were applied for statistical analysis. All data were represented as means ± SD. Independent groups tests were performed using Student’s t-test and one-way ANOVA test. P < 0.05 was considered statistically significant.