With written informed consent, term (>37 weeks gestation) placentas from 40 HIV-1 and Hepatits B seronegative women were obtained immediately following elective caesarian section without labor from Grady Memorial and Emory Midtown Hospitals in Atlanta, GA. Approval of the study was granted from Emory University Institutional Review Board and Grady Research Oversight Committee.
Isolation and Culture of HCs and MDMs
In order to isolate HCs, the decidua basalis was dissected from the placenta. The tissue was thoroughly washed in Hank’s balanced salt solution (HBSS) to minimize peripheral blood contamination and mechanically dispersed in complete medium (RPMI supplemented with 10% FBS, 1 mM L-glutamine, and 1% pen/strep). The minced tissue was re-suspended in complete medium containing 1 mg/ml collagenase IV [Sigma Chemical Co., St. Louis, MO], 10 U/ml dispase [Worthington Biochemical Corp., Lakewood, NJ], and 0.2 mg/ml of DNAse I [Sigma] and incubated in a shaking water bath at 37°C for 1 hour. The digested tissue was washed with PBS and passed through a 70 μm cell strainer [BD Falcon, USA]. The mononuclear cell population was isolated by density gradient centrifugation on Histopaque-1077 [Sigma]. CD14+ Magnetic Cell Sorting was performed using anti-CD14 magnetic beads [Miltenyi Biotech, Germany] as recommended by the manufacturer. In order to isolate MDMs, PBMCs were obtained from the buffy coats of healthy HIV-1 seronegative blood donors by density gradient centrifugation. Isolated cells were washed, re-suspended in RPMI containing pen/strep (1%), glutamine (1%), and heat-inactivated normal human serum (10%) [Mediatech, VA], and seeded into 75-cm2 flasks [Corning Inc., New York]. Non-adherent cells were removed after 2 h of incubation at 37°C. After 24 hours of culture, adherent cells were washed, detached from the flask and counted. Adherent HCs and MDMs were washed with cold EDTA/PBS and detached by incubation with cold EDTA/PBS (2 mM) for 30 min at 4°C. The cells were seeded into 24-well plates and were cultivated for 7 additional days to promote full differentiation into MDMs.
Flow cytometric analysis of cell surface determinants
Cells were labeled with anti-CD14 (F), -CD80 (PE), -CD83 (PE), -CD86 (APC), -HLA-DR (PE), -CCR5 (PerCp-Cy5.5), -CXCR4 (PE-Cy5), -CD4 (APC), and –DC-SIGN (PE) [BD Biosciences, CA]. Briefly, Fc-blocked HCs were incubated with the conjugate antibodies, and unbound antibody washed from the cells. After the final washes, cells were fixed in 1% paraformaldehyde, and cell surface expression was determined by FACS analysis with FACSCanto [Becton Dickinson, CA], and the results were analyzed with the FlowJo software version 8.4.3 [Tree Star, OR].
Infection of HCs and MDMs with HIV-1BaL
The macrophage-tropic isolate, HIV-1BaL was a gift from Jason Hammonds PhD of Emory University School of Medicine. Macrophages were infected at 0.2 TCID50/cell for 4 hours at 37°C. Cells were washed and fresh media added to the cultures. To monitor viral production and particle release, cell supernatants and lysates were collected at days 0, 2, 3, 4, 6, 8, 9 10,, 12, 15, 18 and 21 post-infection. Viral replication and particle release was detected by p24 released into the supernatant and cell-associated p24 by ELISA [Advanced BioScience Laboratories, Inc., MD].
HCs and MDMs were infected at 0.2 TCID50/cell for 4 hours at 37°C. Cells were washed, and fresh media added to the cultures. After 6 days, cells were harvested and mRNA was extracted using the RNAeasy kit (Qiagen, Valencia, CA). The cDNA was transcribed using QuantiTect RT kit (Qiagen). The primer sequences were as follows: forward primer env (5′-GGGGACCAGGGAGAGCATT-3′) and reverse primer env (5′-TGGGTCCCCTCCTGAGGA-3′); forward primer gag (5′- ACATCAAGCAGCCATGCAAAT-3′) and reverse primer gag (5′- ATCTGGCCTGGTGCAATAGG-3′). Real-time PCR was performed using SYBR Green (Qiagen). All reactions were run in triplicate using the Applied Biosystems Prism 7500 Sequence Detection System. Delta-Ct values from the calibrator and experimental groups were measured by subtracting Ct values from target versus the housekeeping transcript, 18S. Each sample is expressed as fold-change relative to HIV-1 infected MDMs.
Cell cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) mitochondrial dehydrogenase assay. Cells were incubated with a 1: 10 dilution of MTT solution to cell media for 20 min at 37 °C. The extent of MTT conversion to formazan by mitochondrial dehydrogenase was determined by measuring optical density at 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The ratio of optical density from treated cells to optical density from control cells reflected the percentage of surviving cells.
Culture of human CBMCs and PBMCs
Cord blood samples were collected from umbilical cords of full-term newborns born to HIV-1 seronegative mothers. PBMCs were obtained from healthy, HIV-1 seronegative adults. CBMCs and PBMCs were separated from heparinized whole-blood samples by density gradient centrifugation on Ficoll-Hypaque [Sigma]. The isolated mononuclear cells were washed in sterile PBS and once in RPMI 1640, maintained in complete medium. CBMCs and PBMCs were stimulated with PHA (5 μg/ml) [Sigma] in the basic medium described above for 24–48 hours prior to infection. Stimulated cells were challenged with HIV-1 immediately following PHA stimulation. Following HIV-1 infection, the PHA-stimulated cells were maintained in complete medium supplemented with IL-2 (100U/ml) [R&D Systems, Minneapolis, MN].
Adherent, HIV-1BaL infected HCs and MDMs were co-cultured with CBMCs or PBMCs for 6 hours. Non-adherent cells were gently removed and washed with PBS, counted and plated in complete medium supplemented with IL-2. Viral replication was detected by p24 release into the supernatant by ELISA.
Cell culture and cytokine measurement
Cells (0.5 x106) were cultured with HIV-1BaL or alone for 2 days. IL-4, IL-6, IL-10, TNF-α, TGF-β, and IFN-γ concentrations were measured by Quantikine ELISA kit [R&D Systems] according to manufacturer’s instructions.
Cytokine treatment and HIV-1 replication
In order to analyze if exogenous IL-4, IL-6, IL-10, TNF-α, TGF-β, and IFN-γ influenced HIV-1 replication, HCs were pretreated for two hours with cytokines [R&D Systems] then inoculated as described earlier with HIV-1BaL. To monitor viral production, cell supernatants were collected at days 0, 3, 6, 9, 12, 15, 18 and 21 post-infection.
HCs and MDMs were re-suspended in RPMI containing 0.1% BSA and 2 x 105 cells, incubated with human CCR5 monoclonal antibodies (5 μg/ml) or along for 15 min – 2 hours, and added to 24-2well Transwell inserts with 8 μm pore polycarbonate membranes (Corning). Inserts were placed in wells containing 600 μl media plus chemoattractants and incubated at 37°C for 18 hours. Cells that migrated through the membrane were stained and counted microscopically in five random high-powered fields. Chemotactic responses are expressed as CI, calculated by dividing the mean number of cells migrating toward chemoattractants by that toward control medium. MIP-1α and MIP-1β were used for chemotaxis at 10 ng/ml (Peprotech, Rocky Hill, NJ).
HIV-1-infected HCs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4 degree C, and then washed with the same buffer. The cells were then post fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for one hour. The cells were subsequently rinsed with de-ionized water, dehydration through an ethanol series ending with absolute ethanol, and then embedded in Eponate 12 resin. Ultrathin sections were cut on a RMC PowerTome XL ultramicrotome at 70 nm, stained with 5% aqueous uranyl acetate and 2% lead citrate, and examined on a JEOL IEM-1400 transmission electron microscope equipped with Gatan UltraScan US1000.894 and Orius SC1000.832 CCD cameras.
Results were analyzed by a one-way analysis of variance (ANOVA). Parametric data were analyzed by using Student’s t test (two-tailed) in order to assess whether the means of two normally distributed groups differed significantly. All non-parametric data was analyzed using Mann–Whitney U Test. All error bars represent the SE.