Parasites and Cell Lines
Murine dendritic cell line DC2.4, human foreskin fibroblast cell line HFF, and murine macrophage cell line RAW264.7 were purchased from the ATCC (American Type Culture Collection, Manassas, USA) and preserved in our laboratory. The DC2.4 cells were cultured in RPMI-1640 (Roswell Park Memorial Institute-1640, bought from Gibco/Invitrogen, Waltham, MA, USA); the HFF and RAW264.7 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, bought from Gibco/Invitrogen). Both of the culture mediums were supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen) and 1% gentamicin (10 mg/mL, Invitrogen, USA). The cells were cultured with 5% CO2 at 37°C. The T. gondii RH strain was propagated in HFF cells in our lab.
Exosome Isolation and Identification
The DC2.4 cells were cultured with RPMI-1640 supplemented with 10% exosome-free FBS (Gibco) in three 75 cm² cell culture flasks to 100% confluence and were infected with RH tachyzoites with a multiplicity of infection of 3 (MOI = 3) for 28 h. Supernatants were collected and exosomes were extracted as described previously [19]. Isolated exosomes were examined using transmission electron microscopy (TEM) (Hitachi, Northeastern Honshu, Japan) at a voltage of 80 kV, as well as the exosomic proteins were detected with Western blotting [19] and the size of exosomes was determined by nanoparticle tracking analysis(NTA) using ZetaView (Particle Metrix, Germany) as previously described [15]. Exosome concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA) as manufacturer’s described.
Western Blot
The isolated exosomes and the transfected cells were collected and lysed using lysis buffer (Beyotime Biotechnology, Shanghai, China), total proteins were loaded for SDS-PAGE, and immunoblotting was performed as described previously [20]. The following primary antibodies were used: rabbit mAb anti-CD9 (1:1000), rabbit mAb anti-TSG101 antibody (1:1000), rabbit mAb anti-HSP70 antibody (1:1000), rabbit mAb anti-iNOS (1:1000), rabbit mAb anti-p65 (1:1000) and rabbit mAb anti-SOCS1 (1:1000) ,which were purchased from Abcam (Massachusetts, USA); mouse mAb anti-GAPDH (1:1000), rabbit mAb anti-phosphorylated IκB alpha (Ser32/Ser36) (1:1000), and rabbit mAb anti-phosphorylated NF-κB p65 (S536) (1:1000), which were purchased from Affinity Biosciences (OH,USA); rabbit mAb anti-IKBα(1:1000) was purchased from GeneTex (Santa Cruz, USA). The secondary antibodies used for the Western blot were HRP goat anti-mouse IgG (1:5000) and HRP goat anti-rabbit IgG (1:5000), which were purchased from abclonal (Wuhan, China).
Uptake of Tg-DC-Exo and DC-Exo by RAW264.7 Cells
RAW264.7 cells were seeded on coverslips in a 12-well plate, approximate 1.1×106 cells per well, and were cultured with DMEM supplemented with 10% exosome-free FBS for 12 h. Exosomes extracted from the supernatants of T. gondii-infected DC2.4 cells (Tg-DC-Exo) and DC2.4 cells (DC-Exo) were labeled with green fluorescence using a PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, USA) according to the experimental procedures described by Lin WC et al. [21]. Briefly, Tg-DC-Exo and DC-Exo (5µg each) were respectively mixed in 40µl of PBS with 50µl Diluent C and 0.25µl PKH67 dye, mixed gently for 5 min at room temperature and 1% bovine serum albumin (BSA) was added to terminate the dying process. PKH67-stained Tg-DC-Exo and DC-Exo were recollected at 100,000×g for 2h at 4°C and resuspended in PBS. PKH67-stained exosomes or the same volume of the PKH67-PBS control were separately added to the RAW264.7 cells and incubated for 6 h. The cells were washed with PBS three times and fixed with 4% paraformaldehyde (Dingguo, China). Subsequently stained with DAPI (Dingguo, China). The coverslips were taken out and then observed under a fluorescence microscope (Nikon, Tokyo, Japan) with a Green Fluorescence Protein (GFP) filter, and images were captured.
Detection of Macrophage Polarization after Treatment of RAW264.7 Cells with Tg-DC-Exo and DC-Exo
The RAW264.7 cells were seeded in 24-well plates, grown overnight to 80% confluence, and then treated with 120 µg/mL of Tg-DC-Exo or DC-Exo for 24 h. The cells were washed with PBS three times and harvested after scraping. Total RNA was extracted to detect the transcription of TNF-α, iNOS, and IL6. The RAW264.7 cells were treated with 120 µg/mL of Tg-DC-Exo or DC-Exo for 48 h. The macrophage M1-type cytokine iNOS and M2-type cytokine Arg1 were detected with a Western blot.
RNA and DNA Isolation and qRT-PCR
The total RNA of exosomes was extracted with an exoRNeasy Serum/Plasma MidiKit (QIAGEN, Duesseldorf, Germany) according to the manufacturer’s protocol. The total RNA of cells was extracted with Trizol reagent (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol. Genomic DNA was removed with a One-Step gDNA Removal kit (Trans Gen Biotech, Beijing, China). For miRNA analysis, exosomic RNA was reverse-transcribed using Super Script™ II Reverse Transcriptase (Thermo Fisher Scientific, Wilmington, USA), and cell RNA was reverse-transcribed using Easy Script® All-in-One First-Strand cDNA Synthesis Super Mix. To evaluate the relative amount of T. gondii tachyzoites, the total DNA of infected cells was extracted with a DNeasy Blood and Tissue Kit and Proteinase K (QIAGEN, Duesseldorf, Germany) according to the manufacturer’s protocol. The specific detection method of the T. gondii B1 gene followed previously described experimental procedures [22]. Real-time polymerase chain reaction (PCR) was performed by using Hieff® qPCR SYBR® Green Master Mix (Yeasen, Shanghai, China) and the Quant Studio™ real-time PCR system (Thermo Fisher Scientific, Wilmington, USA). The primers for quantitative PCR (qPCR) are shown in Table S1. The relative mRNA level was measured with 2−ΔΔCt.
Detection of mir155-5p Transportation to RAW264.7 Cells through Exosome Uptake with a Transwell Experiment
DC2.4 cells were seeded in the upper chambers of 24-well transwell inserts (Corning, New York, USA) and cultured with DMEM supplemented with 10% exosome-free FBS and 1% gentamicin to 100% confluence. The cells were divided into four groups: two groups were left uninfected, and two groups were infected with the T. gondii RH strain (MOI = 3). After infection for 30 min, the culture medium was aspirated and the DC2.4 cells in the upper chambers were washed with PBS three times to remove the unrecruited T. gondii tachyzoites. Meanwhile, RAW264.7 cells were seeded in the lower chambers. The four groups of DC2.4 cells (two infected with T. gondii and two uninfected) in the upper chambers and the RAW264.7 cells in the lower chambers were co-cultured in DMEM supplemented with 10% exosome-free FBS and 1% gentamicin, with or without 100 µL of 10 µm GW4869 (inhibitor of exosome secretion, MedChemExpress), for 52h. Each of these groups was labeled as Normal, DC2.4 + GW4869, DC2.4 + RH, or DC2.4 + RH + GW4869. After that, the RAW264.7 cells were harvested and lysed with lysis buffer (Beyotime Biotechnology, China). The total RNAs were extracted for detection of mir155-5p abundance via quantitative real-time PCR (qRT-PCR). Each group was prepared in triplicate, and the experiment was repeated three times for statistical analysis.
Cell Transfection with Synthesized miRNA or siRNA and Treatment with Exosomes
RAW264.7 cells were seeded in 6-well plates to 80% confluence and were divided into 4 groups. Two groups were transfected with 10 µL of 20 nM mir155-5p mimics and mimic-NC(miRNA mimic-Normal Control), mir155-5p inhibitors and miRNA inhibitors-NC (miRNA inhibitor-Normal Control) (Gene Pharma, Suzhou, China), respectively. The other two groups were transfected with 10 µL of 20 nM si-SOCS1 (siRNA targeting SOCS1) and si-NC (siRNA-Normal Control) (Ribobio, Guangzhou, China), respectively. The transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The exosomes extracted from the cell culture supernatant of the DC2.4 (DC-Exo) and the DC2.4 infected with T. gondii (Tg-DC-Exo) were added to the 80% confluent RAW264.7 cells at 120 ug/well, respectively.
Cell activity, Proliferation, and Polarization Detection
The RAW264.7 cells were incubated with exosomes or transfected with miRNA/siRNA. At 24 h post-treatment, the transcription of the related cytokines was detected via qRT-PCR and Western blotting, and a CCK8 kit (Cell Counting Kit, Trans Gen Biotech, Beijing, China) was used to detect the cell viability and proliferation according to the instructions. In the same way, cell proliferation activity was evaluated by detecting cell absorbance after treatment for 0, 24, 48, 72, and 96 h. Each group was prepared in triplicate, and the experiment was repeated three times for statistical analysis.
Statistical Analysis
The differences between two or three groups were analyzed with Prism (GraphPad Software) using Student’s t-test and one-way analysis of variance (ANOVA). SPSS (version 20) was used to analyze multiple comparisons (Tukey’s), and p < 0.05 indicated that the difference was statistically significant.