Adult BALB/c mice (6–8 weeks old) were bred and maintained in the animal facility of Lovelace Respiratory Research Institute (LRRI). Animal studies were performed in accordance with AAALAC guidelines and approved by the Institutional Animal Care and Use Committee (IACUC). All study amendments and deviations were approved by the IACUC or by the study veterinarian. All experiments were repeated at least three times and each experimental group consisted of two to three mice. Samples were evaluated individually.
2.2. The isolation and culture of bone marrow-derived DCs
Bone marrow (BM) cells were collected and cultured using a protocol modified from that described previously (Inaba et al., 1992
). Briefly, BALB/c mice were anaesthetized and euthanized; the femurs, tibiae, and humerus of mice were removed and cleaned of tissue, and sterilized in 70% ethanol. Both ends of each bone were cut, and marrow was flushed with RPMI-2 (RPMI1640 [Invitrogen, Carlsbad, CA] with 2% heat inactivated fetal Bovine serum [FBS; Hyclone, Logan, UT]). The BM cells were depleted of RBC by ACK lysis buffer (0.15 M NH4
Cl, 1.0 mM KHCO3
, and 0.1 mM EDTA), and filtered (70 μm) to remove large cell aggregates. The remaining cells were washed again, counted by trypan blue exclusion, and resuspended in complete medium ([cRPMI]; 10% FBS, RPMI 1640 medium, 400 mM l
-glutamine, 100 U of penicillin/streptomycin, 5 × 10−7
M 2-mercaptoethanol [2-ME; Invitrogen, Carlsbad, CA]) at a final concentration of 5 × 105
cells/ml. The BM cells were cultured in 6-well plates (2.0 ml/well) with recombinant murine granulocyte macrophage colony stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ) at a final concentration of 10 ng/ml for 3 days. On day 3 of culture, the majority of the non-adherent cells were removed by aspiration of the culture media from each well. Then fresh media containing 10 ng/ml of rmGM-CSF and 10 ng/ml of recombinant murine interlukin-4 (rmIL-4; PeproTech, Rocky Hill, NJ) was added and cultured for another 3 days. On day 6 of culture, the loosely adherent cells, most of which (>80%) are BMDCs as identified by DC markers (CD11c+
MHC class II) using FACS analysis, were harvested using cold Hank's solution. The cells were then washed, resuspended, and counted.
2.3. Preparation of RSV stocks
The A2 strain of RSV was plaque-purified three times under agarose. Following selection, one plaque was used to inoculate a subconfluent HEp-2 cell monolayer. After adsorption for 1 h at room temperature, 10% MEM was added and the infection was allowed to proceed for 3 days at 37°C until the entire monolayer showed cytopathic effects. The contents of the flask were resuspended and distributed in 1 ml aliquots, snap-frozen with alcohol/dry ice, and stored at −80 °C. Virus was derived from this master stock by infecting subconfluent HEp-2 monolayers at multiplicity of infection (MOI) of 0.1, and harvesting the monolayer when it appeared to be completely infected. The cells and media were sonicated on ice and then the suspension was clarified by centrifugation at 1000 × g for 10 min. The supernatant was frozen and stored at −80 °C and thawed rapidly at 37 °C for use. Viral titers were determined by plaque assay. To inactivate RSV, aliquots of the virus were exposed to ultraviolet (UV) light using UV1800 Stratalinker (Stratagene). UV irradiation was titrated to show a lack of infectivity while retaining the ability to visualize RSV proteins by immunoblotting at the appropriate protein size.
2.4. Infection of BMDCs with RSV
BMDCs (1 × 106) were incubated with RSV at different multiplicity of infection (MOI) in 200 μl plain RPMI for 1–2 h at 37°C (viral adsorption phase). Then, cRPMI was added to a total volume of 2 ml and BMDCs were cultured for 6–24 h at 37 °C in 5% CO2. At different time points of infection, cells were collected for subsequent analysis. In some experiments, RSV was inactivated by exposure to UV light prior to inoculation with BMDCs as control.
2.5. RSV RT-PCR
RNA was isolated from BMDCs cultures following RSV- or mock-infection using the RNeasy Kit (Qiagen, Valencia, CA) with optional on-column DNase digestion per the manufacturer's directions. The concentration and quality of RNA was determined by spectrophotometry and equal amounts of RNA were transcribed into cDNA using an Omniscript kit (Qiagen). The mRNA of RSV NS-1 and NS-2 was detected by PCR. The primer sequences for PCR were synthesized by Invitrogen and were as follows:
- RSV NS-1, forward 5′-TTTGGCTAAGGCAGTGA-3′
- reverse 5′-CCATTAGGTTGAGAGCA-3′
- RSV NS-2, forward 5′-ATAATAACATCACTAACCAGAGAC-3′
- reverse, 5′- ATAGTTATGCATAGAGTTGTTGTT-3′
The PCR was cycled 30 times after initial denaturation (94°C, 5 min), with the following parameters: denaturation, 94°C, 45 s; annealing, 52°C, 45 s; and extension, 72°C, 45 s.
2.6. Immunoblotting assays
BMDCs (1 × 106) were cultured in 6-wells plate overnight. Then the cells were mock- or RSV-infected for 24 h prior to subsequent treatment with different cytokines (IFN-γ 100 U/ml for 45 min, IFN-β100 U/ml for 30 min, or IL-10 20 ng/ml for 2 h). BMDC lysates were prepared in lysis buffer (10 mM Tris-HCl, pH 7.5, 15 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, and 0.4 mM PMSF). Protein concentrations were measured using the BCA protein assay kit (Pierce, Rockford, IL) per the manufacturer's directions. Equal amounts of protein samples were resolved on 8–16% SDS–Tris–glycine polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane (Invitrogen). The membrane was blocked with 5% powdered milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated overnight at 4°C with rabbit anti-mouse STAT1 (Cell Signaling, Danvers, MA) diluted 1:1000, rabbit anti-mouse STAT2 (Cell Signaling) diluted 1:1000, rabbit anti-mouse STAT3 (Cell Signaling) diluted 1:1000, rabbit anti-mouse phospho-specific STAT1 (Cell Signaling) diluted 1:1000, rabbit anti-mouse phospho-specific STAT2 (Cell Signaling) diluted 1:1000, rabbit anti-mouse phospho-specific STAT3 (Cell Signaling) diluted 1:1000, or mouse anti-human β-actin (Sigma, St. Louis, MO) diluted 1:20,000 in TBST containing 5% powdered milk. Blots were washed with TBST and incubated with peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG Ab (Pierce) diluted 1:10,000 in TBST containing 1% powdered milk. After washing, blots were developed with a chemiluminescence detection system (Millipore, Billerica, MA).
2.7. Immunofluorescence microscopy
BMDCs were grown and infected with RSV on glass chamber slides (Nalgene Nunc) for 24 h. Following treatment, cells were fixed in 200 μl of 4% paraformaldehyde in PBS for 30 min, washed 3 times with PBS and permeablized using 0.2% Triton X-100 in PBS for 30 min. Following 3 washes with PBS, cells were blocked with PBS containing 10% goat serum (Vector) for 1 h at room temperature, and then incubated with a mouse anti-RSV-F (Serotec) at a dilution of 1:200 and rabbit anti-STAT1, STAT2 at a dilution of 1:100 (Cell Signaling) overnight at 4°C in PBS containing 2% normal goat serum. Primary antibodies were removed and cells were washed 3 times in PBS before addition of secondary antibodies. The nuclei of all cells were stained using 1:400 dilution of Hoescht (Molecular Probes, Invitrogen), while RSV was visualized using a goat anti-mouse Cy5 conjugated antibody (Vector) and STAT proteins were visualized using a goat anti-rabbit Cy3 conjugated antibody (Vector) both at a dilution of 1:200 in 2% goat serum PBS for 1 h at room temperature. Fluorescence microscopy was carried out on a Zeiss Axioplan 2 with an Intelligent Image Innovations CCD Camera using Slidebook software version 184.108.40.206.