The human monocyte cell line, THP-1 (American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotic-antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. To prepare THP-1-derived macrophages, THP-1 cells were incubated with PMA (cat. no. P1585; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 100 ng/ml for 48 h. Following incubation, adherent macrophages were maintained in complete medium at 37°C with 5% CO₂.

A. fumigatus A1 was kindly provided by the Microbiological Laboratory of Jinling Hospital (Nanjing, China). A. fumigatus was cultured on Sabouraud dextrose agar (Thermo Fisher Scientific, Inc.) containing penicillin (100 U/ml) and streptomycin (100 µg/ml; Gibco; Thermo Fisher Scientific, Inc.) for 5 days at 37°C.

Conidia were washed from agar plates using 5 ml sterile phosphate-buffered saline (PBS) supplemented with 0.025% Tween-20. The suspension was filtered through nylon filters with 25-µm pores to separate the conidia from the contaminating mycelium. Resting conidia were diluted in PBS and counted using a hemocytometer. To prepare fluorescein isothiocyanate (FITC)-labeled conidia, a total of 5×10⁸/ml conidia were suspended in 0.1 M carbonate buffer (pH 9.0) prior to incubation with FITC (cat. no. F-4274; Sigma-Aldrich; Merck KGaA) at a final concentration of 0.16 mg/ml overnight at 4°C. The suspension was then diluted and washed twice with PBS. FITC-labeled conidia were diluted in PBS to the desired concentration (4×10⁸/ml) and stored at 4°C in the dark (3,19).

The PU.1 recombinant adenovirus vector used in the study was provided by Shanghai R&S Biotechnology Co., Ltd. (Shanghai, China) and the final titer was 8×10¹¹ IU/ml. THP-1 derived macrophages were seeded in six-well plates (Corning Incorporated, Corning, NY, USA) at a density of 8×10⁵ cells/well. Cells were divided into the following three groups: Mock untransduced group; an empty-vector transduction group (Ad group), where cells were transduced at a multiplicity of infection (MOI) of 1,500; and a PU.1 recombinant adenovirus vector-transduced group (Ad-PU.1), where were transduced at an MOI of 1,500. Following co-culture in 1 ml of RPMI 1640 medium for 12 h, the adenovirus-containing medium was removed and replaced with complete RPMI 1640 medium. Total RNA and protein were extracted at 48 h post-transduction and stored at −80°C until required.

Small interfering RNA (siRNA) targeting PU.1 and negative control siRNA were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequence for the PU.1 siRNA was as follows: Forward, 5′-UAUAGAUCCGUGUCAUAGGGCACCA-3′, and reverse, 5′-UGGUGCCCUAUGACACGGAUCUAUA-3′. THP-1-derived macrophages were seeded in six-well plates (Corning Incorporated) at a density of 8×10⁵ cells/well. For each transfection, 160 pmol PU.1 siRNA (siPU.1 group) or negative control siRNA (siNC group) was mixed with 8 µl Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 500 µl Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.), and incubated at room temperature for 20 min. The mixture was then added to each well containing the cells and the plate was incubated for 8 h at 37°C. Cells that were untransfected were used as a mock transfection group. Following incubation, 1.5 ml complete RPMI 1640 medium was added to each well. Total RNA and protein were extracted at 48 h post-transfection and stored at −80°C until required.

Following overexpression or silencing of PU.1, THP-1-derived macrophages were stimulated with A. fumigatus resting conidia (MOI=1, if not indicated otherwise) for 0, 4, 8 and 12 h, respectively. Total RNA and culture supernatants were then isolated at these indicated time points. Samples were stored at −80°C until required.

Cells were lysed with radioimmunoprecipitation assay lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40 and 1% sodium deoxycholate with 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration was determined using a bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology, Hangzhou, China) according to the manufacturer's protocol. Protein lysates (10 µg) were separated using 10% SDS-polyacrylamide gels (cat. no. P0012A; Beyotime Institute of Biotechnology) and transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were then blocked with 5% skimmed milk (Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. The membranes were subsequently hybridized with primary rabbit anti-human polyclonal antibodies against PU.1 (cat. no. 2258; Cell Signaling Technology, Inc., Danvers, MA, US), Dectin-1 (cat. no. 9051; Cell Signaling Technology, Inc.), TLR-2 (cat. no. 2229S; Cell Signaling Technology, Inc.) and TLR-4 (cat. no. sc-10741; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:1,000 dilution overnight at 4°C. Membranes were also incubated with primary rabbit anti-human polyclonal antibodies against β-tubulin (cat. no. 2146S; Cell Signaling Technology, Inc.) as a loading control for PU.1, and β-actin (cat. no. 12620; Cell Signaling Technology, Inc.) for Dectin-1, TLR-2 and TLR-4, at a 1:1,000 dilution overnight at 4°C. Following incubation with the relevant horseradish peroxidase-conjugated polyclonal goat anti-rabbit secondary antibody (cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at 1:5,000 dilution at room temperature for 2 h, an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) was used for detection of immunoreactive proteins. Images of protein bands were acquired using the G:BOX Chemi XR5 system (Syngene, Frederick, MD, USA) (20). All antibodies were diluted in Tris-buffered saline with 0.1% Tween-20.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The concentration and purity of extracted RNA were (260/280 ratio) was determined using a NanoDrop^™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized from total RNA (1 µg) using PrimeScript RT Master Mix (cat. no. RR036A; Takara Biotechnology Co., Ltd., Dalian, China). The sequences of human-specific primers were synthesized by GenScript (Nanjing, China) and are listed in Table I. qPCR was performed using an Applied Biosystems Prism 7500 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR^® Premix Ex Taq^™ (cat. no. RR420A; Takara Biotechnology Co., Ltd.). PCR was performed as follows: Denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Relative mRNA expression levels were normalized to GAPDH, and calculated using the 2^−∆∆Cq method (21).

TNF-α and interleukin (IL)-10 levels in the cell culture supernatants were determined using commercially available Quantikine ELISA kits (cat. nos. DTA00C and D1000B, respectively; R&D Systems, Minneapolis, MN, USA), strictly according to the manufacturer's protocols. Absorbance was recorded at 450 nm in a microplate reader (Thermomax; Molecular Devices, LLC, Sunnyvale, CA, USA).

THP-1-derived macrophages were seeded on 22×22 mm coverslips in six-well plates (Corning Incorporated) at a density of 8×10⁵ cells/well. Following transfection with PU.1 siRNA or transduction with Ad-PU.1 for 48 h, cells were co-cultured with 4×10⁶ FITC-labeled resting conidia (MOI=5) in 1 ml complete RPMI 1640 medium at 37°C and 5% CO₂ for 4 h. Cells were then washed vigorously three times with PBS, and fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were gently washed three times with PBS and incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; cat. no. C1036; dilution, 1:500; Beyotime Institute of Biotechnology) and DAPI (cat. no. C1002; dilution, 1:1,000; Beyotime Institute of Biotechnology) to stain the membrane and nucleus, respectively. Finally, the coverslips were removed, gently washed three times with PBS and mounted onto glass slides using SlowFade Gold antifade reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A confocal laser-scanning microscope (Fluoview^® FV10i; Olympus Corporation, Tokyo, Japan) with a ×63 objective oil immersion lens was used to obtain all images. FITC-labeled resting conidia, cell membranes and nuclei were observed by the fluorescence intensities of FITC (green; excitation, 495 nm; emission, 525 nm), DiI (red; excitation, 549 nm; emission range collected, 565 nm) and DAPI (blue; excitation, 364 nm; emission, 454 nm).

THP-1-derived macrophages (8×10⁵ cells/well) were exposed to resting conidia (MOI=0.5) in six-well plates for 2 h. Killing experiments were performed as previously described (3). Briefly, non-adherent cells and non-phagocytosed conidia were removed by washing the cells with pre-warmed PBS, and fresh medium was added (0 h time point). The cells were subsequently incubated for 4 or 8 h. At 0, 4 and 8 h time points, the plates were frozen at −80°C, and immediately thawed at 37°C to lyse the cells and harvest the conidia. Serial dilutions were performed in sterile medium, and solutions were used to inoculate Sabouraud dextrose agar (Thermo Fisher Scientific, Inc.). Colonies were counted following incubation for 24 h at 37°C. The percentage of killed conidia was calculated by dividing the difference between the number of conidia recovered at 0 and 4 or 8 h by the total number of conidia recovered at 0 h, and then multiplying by 100 (22).

Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) and the SPSS statistical analysis software program (version, 19.0; IBM SPSS, Armonk, NY, USA). All experiments were performed in triplicate. Data are presented as the mean ± standard deviation. One-way analysis of variance followed by a Bonferroni post hoc test was used to compare differences among three groups when equal variances were assumed, and a Dunnett's T3 multiple comparisons test was used when equal variances were not assumed. P<0.05 was considered to indicate a statistically significant difference.

A recombinant adenoviral vector carrying the PU.1 gene SPI1 (Ad-PU.1) was provided by Shanghai R&S Biotechnology Co., Ltd. and used to transduce THP-1 derived macrophages. RT-qPCR analysis demonstrated that PU.1 mRNA increased by 80.4-fold in the Ad-PU.1 group when compared with the Mock group (P<0.05; Fig. 1A). Western blot analysis confirmed the results of the RT-qPCR analysis (Fig. 1B). In addition, RT-qPCR analysis demonstrated that PU.1 mRNA levels were reduced to 19.0% of the Mock group following transfection with siPU.1 (P<0.05; Fig. 1C). Consistent with these observations, western blotting revealed that PU.1 protein levels were markedly decreased in the siPU.1-transfected group when compared with the mock group (Fig. 1D). The results demonstrated that PU.1 was successfully overexpressed and silenced in THP-1-derived macrophages.

As PU.1 is essential for the transcriptional regulation of Dectin-1, TLR-2 and TLR-4 (13–18), the mRNA and protein expression levels of these genes in THP-1-derived macrophages following transduction with Ad-PU.1 and transfection with siPU.1 were analyzed. When compared with the mock group, Dectin-1, TLR-2 and TLR-4 mRNA expression increased by 8.31, 2.56 and 4.28-fold, respectively (Fig. 2A). Similarly, western blot analysis revealed that the protein expression levels of these three receptors increased when compared with the mock group (Fig. 2B). Conversely, the siPU.1 group exhibited a significant reduction in Dectin-1, TLR-2 and TLR-4 mRNA expression levels by 4.64, 4.04 and 4.33-fold, respectively, when compared with the mock controls (Fig. 2C). Consistent with these results, the protein expression levels of these factors were decreased in the siPU.1 group when compared with the mock group (Fig. 2D). The results indicate that PU.1 overexpression led to upregulation of Dectin-1, TLR-2 and TLR-4, and silencing of PU.1 led to downregulation of Dectin-1, TLR-2 and TLR-4.

In order to investigate whether overexpression of PU.1 promotes immune defense against A. fumigatus, the cytokine release, phagocytosis and killing ability of THP-1-derived macrophages was examined. Cells were stimulated for 0, 4, 8 and 12 h with resting conidia (MOI=1) at 48 h following transduction with Ad-PU.1. The mRNA levels of TNF-α and IL-1β increased in a time-dependent manner in the Ad-PU.1 group, whereas, IL-10 exhibited no significant increase in expression (Fig. 3A-C). The protein expression patterns of TNF-α and IL-10 in cell culture supernatants were consistent with the observed mRNA expression patterns (Fig. 3D and E).

AMs constitute the first line of a host's immune defense, and facilitate the initiation of the innate immune response to A. fumigatus infections, including phagocytosis and killing (23,24). Therefore, the phagocytosis and killing ability of THP-1-derived macrophages following overexpression of PU.1 was examined. Cells were stimulated with resting conidia (MOI=0.5) for 4 and 8 h, respectively. The killing rate in the Ad-PU.1 group was significantly higher when compared with the control groups (Fig. 3F). For the analysis of phagocytosis, cells were stimulated with FITC-labeled resting conidia (MOI=5) for 4 h. The confocal laser-scanning microscopy results shown in Fig. 3G, demonstrated that phagocytosis was enhanced following overexpression of PU.1. The results therefore suggest that overexpression of PU.1 promoted cytokine release, and enhanced phagocytosis and the killing ability of THP-1-derived macrophages during A. fumigatus conidia stimulation.

As overexpression of PU.1 appears to enhance the immune defense against A. fumigatus, the authors of the present study investigated whether silencing of PU.1 expression was associated with decreased cytokine release, phagocytosis and a reduced killing ability of THP-1-derived macrophages. Cells were stimulated for 0, 4, 8 and 12 h with resting conidia (MOI=1) at 48 h following transfection with PU.1 siRNA. The mRNA levels of TNF-α and IL-1β decreased, while IL-10 levels increased significantly in the siPU-1 group when compared with the mock group (Fig. 4A-C). Similar results were observed for the protein levels of TNF-α and IL-10 in culture supernatants (Fig. 4D and E). To determine the killing ability, cells were stimulated with resting conidia (MOI=0.5) for 4 and 8 h. As shown in Fig. 4F, the killing rate in siPU.1 group was significantly lower compared with the control groups at 4 h; however, no significant difference at 8 h was observed (Fig. 4F). Following the stimulation of cells with FITC-labeled resting conidia (MOI=5) for 4 h, a decrease in phagocytosis was observed in the siPU.1 group when compared with the controls (Fig. 4G). These results demonstrate that silencing of PU.1 decreased cytokine release, phagocytosis and the killing ability of THP-1-derived macrophages during A. fumigatus conidia stimulation.