The human extravillous trophoblast cell line SGHPL-4 (kindly provided by Drs. Guy Whitley and Judith Cartwright, St. George’s University, London, England) was used for these studies. This cell line was derived from first-trimester chorionic villous tissue immortalized with SV40 large T antigen. These cells have been shown to share many characteristics with first-trimester primary EVT, including expression of cytokeratin-7, HLA-G, hCG, and hPL when cultured on an artificial extracellular matrix (Matrigel)
. These cells are permissive for the full replication cycle of human cytomegalovirus
[13, 44]. SGHPL-4 cells were cultured in Ham’s F10 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycin (all from Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. For serum starvation, the media was replaced with Ham’s F10 prepared as above but with reduced FBS (0.5%).
HCMV was propagated in human foreskin fibroblast cells (HFF; ATCC, Manassas, VA). These cells were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycin (all from Invitrogen) at 37°C in 5% CO2.
Viral strains and propagation
Several viral strains were used for these studies. Towne-GFP (an attenuated but well-characterized laboratory isolate) and TRpM1a (a clinical isolate) were kindly provided by Dr. Dan Streblow (Oregon Health Sciences University, Beaverton, OR). Other viral strains (Toledo and Towne) were purchased from ATCC. Total virus was prepared from cell culture supernatants of infected HFFs as previously described
. Briefly, infected HFFs were incubated at 37°C in 5% CO2 in DMEM containing 10% FBS until 100% of the cells demonstrated cytopathic effect (CPE). Regular feeding of the cells was then ceased and the cells were incubated for an additional 4–5 days, at which point the cells were easily detached from the plastic. Supernatants were collected and clarified by centrifugation for 10 minutes at 2500 rpm at 4°C. The virus was aliquoted and frozen at −80°C and then stored in liquid nitrogen. Viral titers were calculated by staining for the immediate early antigens of HCMV 24 hours post-infection.
All HCMV infections were carried out as follows. Briefly, SGHPL-4 cells were seeded on tissue culture plates and allowed to adhere for at least 24 hours at 37°C. The cells were then washed with 1X Dulbecco’s phosphate buffered saline (DPBS, Invitrogen) and serum-starved in Ham’s F10 medium containing 0.5% FBS for an additional 24 hours. HCMV was diluted in serum-free Ham’s F10 in a minimal volume for the surface area to be infected. The cells were incubated with virus at 37°C for 90 minutes with occasional rocking. Unbound virus was removed by aspiration and fresh Ham’s F10 containing 0.5% FBS was added to the infected cells. For each experiment, control cells were mock-infected using equivalent volumes of media.
HCMV was UV-inactivated as previously described
. Briefly, virus thawed on ice was spread onto a 10-cm plate and cross-linked four times at 426 mJ, with gentle mixing after each cycle. The virus was then diluted and used for infections as described above.
Antibodies and reagents
For immunofluorescence, the following primary antibodies were used: mouse anti-human CXCL12 (R&D Systems, Minneapolis, MN, MAB350). Detection of HCMV immediate early (IE) gene expression was performed using mouse anti-human cytomegalovirus IE 1/2 (Millipore, Billerica, MA, MAB810). Isotype-matched secondary antibodies conjugated to either Alexa-488, Alexa-555, or Alexa-594 (Invitrogen) were used as indicated. For flow cytometric analysis of chemokine receptor expression, mouse anti-human CXCR4 (eBioscience, San Diego, CA, 12–9999) and mouse anti-human CXCR7 (Biolegend, San Diego, CA, 331104) antibodies directly conjugated to phycoerythritin (PE) were used.
For Western blotting, the following antibodies were used: rabbit anti-human CXCR4 (Abcam, ab2074), rabbit anti-human CXCR7 (Abcam, ab72100), and rabbit anti-human beta actin (Abcam, ab8227). Appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) were used for detection (1:5000, KPL, Gaithersburg, MD).
Recombinant CXCL12 was purchased from R&D Systems and reconstituted in DPBS supplemented with 0.1% bovine serum albumin. Neutralization of CXCR4 activity was obtained with mouse anti-human CXCR4 antibody (clone 12G5, Abcam ab21555) diluted to a final concentration of 10 μg/ml in serum-free Ham’s F10 medium.
SGHPL-4 cells were infected with HCMV at an MOI of 1 and supernatants were harvested at appropriate timepoints post-infection. The supernatants were spun at 2500 rpm to pellet any cellular debris, aliquoted, and frozen at −80°C prior to analysis.
Secretion of CXCL12 into cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA, R&D Systems, SDF-1α Quantikine Immunoassay kit) per the manufacturer’s instructions. ELISA plate absorbance was read at 450 nm with correction at 540 nm using a μQuant Universal Microplate Spectrophotometer (BioTek Instruments; Winooski, VT). The data were normalized to a standard curve prepared with serial dilutions of recombinant CXCL12.
SGHPL-4 cells were plated on 4-well chamber slides coated with collagen type IV (BD Biosciences), and then mock-infected or infected with HCMV as outlined above. At appropriate timepoints, the cells were fixed with 2% methanol free paraformaldehyde (Ted Pella, Inc. Redding, CA) for 20 minutes at room temperature. Free aldehydes were quenched by incubation with 50mM NH4Cl for 10 minutes, and the cells were permeabilized with 0.1% Triton-X 100 in DPBS for 8 minutes. The slides were blocked with DPBS supplemented with 5% BSA and 0.05% Triton-X 100 for 1 hour at room temperature. Primary antibodies were diluted in DPBS supplemented with 1% BSA and 0.05% Triton-X 100 for 1 hour. After extensive washing, secondary antibodies conjugated to Alexa-488, Alexa-555, or Alexa-594 (Invitrogen) were added and nuclei were counterstained with 4′,6-diamidino-2-phenylindole,dihydrocholoride (DAPI, Invitrogen). The slides were then washed with DPBS and mounted with Prolong Antifade mounting medium (Invitrogen) and covered with glass coverslips. For each experiment, wells stained with secondary antibodies alone were included as controls for background fluorescence. Slides were imaged using a Zeiss Axio Plan II microscope (Thornwood, NY) and images were deconvolved using SlideBook 5.0 Intelligent Imaging Software (Intelligent Imaging Innovations; Denver, CO).
Cell surface expression of CXCR4 and CXCR7 was analyzed by flow cytometry. SGHPL-4 cells grown in 6-well plates were serum-starved for 24 hours at 37°C. The cells were then infected with Towne HCMV at an MOI of 1 and incubated for an additional 24, 48, 72, or 96 hours. The samples were blocked in DPBS containing 2% FBS for 20 minutes on ice prior to incubation in blocking solution containing antibodies to CXCR4 and CXCR7 directly conjugated to PE for 30 minutes on ice in the dark. After washing, the samples were fixed in 4% methanol-free formaldehyde (Ted Pella, Inc.) for 20 minutes in the dark, and then washed with DPBS. The samples were stored in DPBS containing 0.1% BSA and 0.1% NaN3 at 4°C until they were run on the flow cytometer. The samples were analyzed using a BD FACSCalibur on CellQuest Pro software (BD Biosciences, San Jose, CA). Unstained control cells and cells stained with PE-conjugated isotype controls were used to set the acquisition controls and voltages. The data were analyzed using CellQuest Pro software (BD Biosciences).
SGHPL-4 cells were plated on 10-cm plates and grown to near confluency, then serum-starved in Ham’s F10 containing 0.5% FBS for 24 hours. For long-term HCMV infection studies, the cells were infected with HCMV at MOI of 1 for 90 minutes at 37°C, at which point unbound virus was removed and the cells were incubated in Ham’s F10 containing 0.5% FBS for 24–96 hours. Mock-infected controls were included for each time point.
Protein lysates were harvested as previously described
[13, 45]. The plates were washed in cold DPBS and the cells were lysed with RIPA buffer containing a proteinase inhibitor cocktail (PIC), two phosphatase inhibitors (PhIC I and II) and 1mM phenylmethanesulfonylfluoride (PMSF; all from Sigma-Aldrich; St. Louis, MI). The lysates were sheared and. cellular debris was removed. Total protein concentrations were estimated with the Micro BCA protein assay kit (Thermo Fisher; Rockford, IL).
Total protein (25 μg per sample) were separated on 4-12% NuPage SDS-polyacrylamide gels (Invitrogen). Proteins were transferred by blotting to PVDF membranes (Invitrogen) and were blocked in 5% non-fat dry milk (NFDM) in Tris-buffered saline containing 0.1% Tween-20 (TBST) prior to incubation with primary antibody overnight at 4°C. After extensive washing, the blots were incubated with secondary antibodies (goat anti-mouse IgG, rabbit anti-goat IgG or goat anti-rabbit IgG, 1:5000, KPL Protein Research Products) for 1 hour at room temperature. Antigen-antibody complexes were detected using an enhanced chemiluminescence system (Thermo Fisher) and digital images of blots were acquired using an LAS4000 imager (Kodak, Rochester, NY). Blots were subsequently stripped in Restore Plus Western Blot Stripping Buffer (Thermo Fisher) and reprobed using a rabbit polyclonal antibody directed against human β-actin (Abcam) as a loading control.
Quantitative real-time PCR
Total RNA was isolated from serum-starved SGHPL-4 cells that had been mock-infected, infected with UV-inactivated HCMV, or infected with the Towne HCMV strain. RNA was isolated using the Qiagen RNeasy Mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). Isolated RNA was treated with DNase I (Ambion, Inc.; Austin, TX) and concentration was measured using a NanoDrop Spectrophotometer (Thermo Scientific). Total RNA (1 μg) was reverse transcribed with iScript cDNA Synthesis Kit (Biorad; Hercules, CA) as per the manufacturer’s instructions.
Quantitative real-time PCR was performed using the iCycler Real-Time PCR Detection System (BioRad). Specific primer pairs used in these aims are listed in Table
1. PCR cycling conditions were as follows: 1 denaturing step at 95°C for 3 minutes, 40 cycles of 95°C for 15 seconds followed by 60°C for 30 seconds, 1 cycle at 95°C for 1 minute, and 1 cycle of 60°C for 1 minute. Following the PCR reaction, melt curve reactions were performed to confirm the specificity of each primer pair and exclude the presence of primer dimers from the reaction. The melt curve was performed via 80 cycles beginning at 55°C and incrementally increasing by 0.5°C every 30 seconds. Relative quantitation was determined using the comparative CT method with data normalized to 36B4 and calibrated to the average CT of mock-infected control at the specified time point.
Migration and invasion assays
Cellular migration and invasion were determined using the quantitative FluoroBlok assay as previously described
. Serum-starved SGHPL-4 cells were mock- infected or infected with Towne HCMV as described above. 24 hours post-infection, the cells were trypsinized and loaded into FluoroBlok inserts with 8 μm pores (BD Biosciences). To assess the role of CXCR4 on migration and invasion toward CXCL12, some samples were pre-treated with 12G5 (a neutralizing antibody against CXCR4) or a mouse IgG isotype control, both at 10 μg/ml, for 30 minutes prior to the beginning of the migration or invasion assay.
For invasion assays, the inserts were pre-coated with growth factor reduced Matrigel (BD Biosciences) diluted 1:10 in serum-free Ham’s F10 and the Matrigel was allowed to solidify for 1 hour at 37°C. For migration assays, the inserts were left uncoated. Each well was loaded with 2.5x105 cells in a total volume of 200 μl of serum-free medium. The lower wells of the chamber were loaded with serum-free culture medium containing varied doses of recombinant CXCL12. Human EGF (10 ng/ml) was used as a positive control for all assays and serum-free medium alone was used as a negative control. Invasion assays were allowed to proceed for 24 hours whereas migration assays were incubated for 6 hours.
At the end of the experimental time period, the plates were removed from the incubator and any cells remaining on top of the insert were removed by aspiration. The cells on the lower surface of the inserts were then fluorescently labeled with Calcein AM (Invitrogen). The plates were then incubated at 37°C for an additional hour, which allowed for fluorescent labeling of the invasive cells on the lower surface of the membrane. Four 10X pictures of each well were taken using a Nikon TE300 inverted epifluorescent microscope (Olympus Optical Company, Lewisville, TX) and the mean fluorescence per image was determined using ImageJ software (NIH, Bethesda, MD).
The data are presented as mean ± standard error of the mean
. Data were compared to negative control samples and each other by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons post hoc test (GraphPad Prism, Version 5, La Jolla, CA). For experiments with only two groups, data were analyzed by one-tailed unpaired t test. Statistical significance is denoted on figures with asterisks (*; p<0.05, **; p<0.01, ***; p<0.001).