Objective
Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection?
Study Design
Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction.
Results
Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium ( P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes .
Conclusion
Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.
Beyond providing a physical barrier between the maternal and fetal vasculature, the placenta governs the exchange of gases, nutrients, and waste products between these 2 compartments. In the hemochorial placenta, this exchange is regulated primarily by the syncytiotrophoblasts, a layer of multinucleated, terminally differentiated cells that are bathed in the maternal blood and play a critical role in protecting the developing fetus from invading pathogens. Despite this defensive barrier, some pathogens are able to invade the fetal environment.
Viral infection of the intrauterine compartment can spread to the fetus and/or the mother. Active maternal viral infections can lead to infection during delivery or to pregnancy loss (either early or late) resulting from systemic spread of the infection. Viruses that are transmitted directly to the fetus can result in developmental abnormalities or fetal or neonatal disease. For example, prior to the widespread use of vaccination, fetal infection rates by the rubella virus were nearly 50% during maternal rubella infection in the first trimester of pregnancy and were associated with congenital rubella syndrome, characterized by deafness, cataracts, damage to the central nervous system, and cardiac defects. Congenital infection with varicella during pregnancy can lead to spontaneous abortion or neonatal varicella infection, which may result in devastating birth defects known as congenital varicella syndrome. Venezuelan equine encephalitis virus (VEEV) has been linked to pregnancy complications such as spontaneous abortion. Human immunodeficiency virus (HIV-1) tends to be transmitted during vaginal delivery or invasive procedures.
Beyond the formation of a syncytial physical barrier, mechanisms by which placental trophoblasts influence viral infections are insufficiently understood. We recently demonstrated that primary human trophoblasts (PHT) are resistant to infection by an unrelated panel of viruses. Furthermore, viral resistance was conferred to nontrophoblast cells when incubated with conditioned medium from PHT cells. This resistance was mediated, at least in part, by exosomal delivery of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC), which is the largest miRNA cluster in human beings unique to primates and almost exclusively expressed in the placenta. C19MC miRNAs are highly expressed in exosomes released from PHT cells, and can be found circulating in the plasma of pregnant women. In cells exposed to PHT-conditioned medium we also observed a strong induction of autophagy, a prosurvival catabolic process where cellular organelles are partly or fully enclosed in cytoplasmic phagosomes, and degraded upon fusion with the lysosomes. Autophagy was also observed in cells transfected with selected miRNA members of the C19MC, and attenuation of autophagy mitigated this antiviral effect. Here we expand upon our previous observations and focus on viruses that are pertinent to fetal infection during pregnancy and/or delivery, including rubella virus and other togaviruses, HIV-1, and varicella zoster virus (VZV). Additionally, we compared these effects to infection by 2 clinically relevant nonviral perinatal pathogens, Toxoplasma gondii and Listeria monocytogenes .
Materials and Methods
Study design
To assess the ability of human trophoblasts to confer pathogen resistance to nontrophoblastic cells, we collected conditioned medium from PHT cells, cultured from 48-72 hours, or control nonconditioned medium. This medium was added to nontrophoblast recipient cells for 24 hours prior to infection. Pathogens were then added to cells exposed to either medium, and subsequent infection was assessed utilizing the assays listed below. Infection was quantified relative to control conditions. This design is illustrated in Figure 1 .
Cells
Human osteosarcoma U2OS, human foreskin fibroblast (HFF), melanoma-derived cells (MeWo), and TZM-bl cells were cultured in Dulbecco modified Eagle medium ( DMEM; Corning, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO) and antibiotics. Human epithelial cells (Caco-2 cells, ATCC [Manassas, VA] HTB-37) were cultured in Eagle minimal essential medium (MEM) containing 20% heat-inactivated FBS and 10 U penicillin and streptomycin. Vero African green monkey kidney cell line, commonly used for assessment of viral infections, were maintained in DMEM supplemented with 5% FBS and antibiotics.
PHT cells were acquired from healthy singleton term placentas, using the procedure previously described, with modifications. Cells were maintained in DMEM containing 10% bovine growth serum (HyClone, Logan, UT), 20 mmol/L HEPES, and antibiotics at 37°C. Cells were maintained 72 hours after plating, with cell quality monitored morphologically and by human chorionic gonadotropin levels (enzyme-linked immunosorbent assay; DRG International, Mountainside, NJ) in the medium, which show a characteristic increase as cytotrophoblasts differentiate into syncytiotrophoblasts.
Conditioned medium preparation
Conditioned medium samples were collected from PHT cultures as previously described, and only medium that demonstrated at least 70% reduction in vesicular stomatitis virus (VSV) infection was used for subsequent infectivity assays. Briefly, U2OS cells were exposed to conditioned or nonconditioned PHT medium for 24 hours, and infected with VSV at a multiplicity of infection (MOI) of 1 for 6 hours or until cytopathic effect was evident. Cells were then lysed with 1 mL of Qiazol lysis reagent (Qiagen Sciences, Germantown, MD), and infection was quantified by reverse transcription quantitative polymerase chain reaction, as previously described. Notably, 65% of the media screened exhibited at least 70% reduction in VSV infection, and thus used for subsequent assays.
Rubella plaque assays
Vero cells were preexposed to either conditioned or nonconditioned medium and infected with rubella virus at an MOI of 10. Plaque assays were performed with serial dilution of the virus. A total of 1 mL of each dilution and a control (phosphate buffered saline [PBS]/1% FBS) were plated on 30-mm plates confluent with Vero cells, and the plates were incubated for 1 hour at 37°C. Cells were overlaid with a liquid agar solution (60 mL 0.4% liquid agar, 34 mL 3X MEM, 1 mL FBS, 3 mL 5% NaHCO 3 , 0.1 mL penicillin/streptomycin, and 0.1 mL diethylaminoethanol). The plates were incubated for 7 days at 37°C. After incubation, the agar was removed, and the plates were stained with crystal violet solution to reveal plaques. Duplicate plaque assays were performed for each infection, and the final titer of each infection was the average of the 2 plaque assays.
Alphavirus luciferase assays
Vero cells were preexposed to either conditioned or nonconditioned medium for 24 hours prior to infection. Reporter alphaviruses that expressed a cleavable firefly luciferase in between the capsid and PE2 proteins (as described ) were added to the cells at an MOI of 0.1 for 8 hours and lysed with 1X passive lysis buffer (Promega, Madison, WI). Infection was quantified by measuring luciferase expression and normalized to protein level. Alphavirus constructs included eastern equine encephalitis virus (EEEV), VEEV, chikungunya virus (CHIKV), and Sindbis virus (SINV). These virus constructs were also used to infect U2OS cells transfected with miRNA mimics from the C19MC, as described below. These experiments were conducted with an MOI of 1 for EEEV, VEEV, and CHIKV while SINV was used at an MOI of 0.1.
HIV infectivity assay
Utilizing the TZM-bl cell line, a HeLa cell derivative that is CD4 + and expresses CCR5 and CXCR4, as well as a trans-activator of transcription-inducible luciferase reporter, HIV-1 infectivity was quantified on the basis of relative luciferase expression. These cells, which are permissive to wild type HIV-1, were preexposed to PHT-conditioned medium or nonconditioned medium for 24 hours prior to infection. HIV-1 NL4-3 was added at increasing concentrations, indicated in Figure 3 . After 48 hours, infected cells were washed with PBS and lysed in luciferase lysis buffer (Promega). Lysates (40 μL) were transferred to white 96-well plates, and 50 μL of luciferase reagent (Promega) was injected into each well. Luciferase activity was determined by detection of luminescence recorded by Synergy 2 SL luminescence microplate reader (BioTek, Winooski, VT).
VZV luciferase assays
VZV infectivity was measured using reporter viruses expressing firefly or Renilla luciferase reporter enzymes, respectively. Recombinant viruses were developed from a VZV bacterial artificial chromosome using methods detailed previously. The luciferase gene was placed directly downstream of either the immediate early gene encoding the latency-associated regulatory protein IE63 or the late VZV open reading frame (ORF) 9 gene encoding the abundant VZV tegument protein at the native locus, and was expressed as bicistronic messenger RNA with a T2A ribosome skipping motif (MB Yee and PR Kinchington, personal communication). VZV permissive HFF cells were preexposed to conditioned or nonconditioned medium for 24 hours. Cells were then infected with 1000 pfu/mL of either VZVORF9Luc or VZVORF63Luc vector for 48 hours. Cells were lysed, and luciferase expression was quantified as described above.
Fluorescence microscopy
T gondii infection was quantified using a yellow fluorescent protein (YFP)-tagged RH strain provided by David Roos, Department of Biology, University of Pennsylvania (Philadelphia, PA), as previously described. HFF cell monolayers were cultured in 8-well chamber slides (Nunc Lab-Tek; Thermo-Fisher, Waltham, MA) at 37°C, 5% carbon dioxide. Either conditioned or nonconditioned medium was added to the cells 24 hours prior to infection. T gondii RH-YFP was added at an MOI of 0.5. At 48 hours postinfection, cells were washed and fixed with 4% paraformaldehyde in PBS and permeabilized with 0.25% Triton X-100 in PBS. Fixed monolayers were then mounted with Vectashield (Vector Laboratories, Burlingame, CA) containing DAPI. Images were captured with an Olympus FluoView 1000 laser scanning confocal microscope. Parasitophorous vacuole number and size, measured by region of interest length (μm), were quantified using Image J software (National Institutes of Health).
Listeria infectivity assay
Wild type L monocytogenes (DP10403S) was cultured overnight at 37°C in brain and heart infusion (BHI). The following day, bacteria were diluted (1/20) in BHI and grown at 37°C until optic density 600 nm reached 0.7–0.8. Bacteria were washed 3 times with PBS and suspended in MEM at the indicated MOI. Caco-2 cells were plated in 24-well plates at a density of 40,000 cells/well on glass coverslips coated with rat tail collagen. After 24 hours, the cell culture medium was replaced with 500 μL conditioned or control medium, in triplicate, for 24 hours. Caco-2 cells were washed with MEM and infected with L monocytogenes at the indicated MOI. The bacterial suspension (500 μL MEM) was added to each well, and the cell culture plate was centrifuged at 1500 rpm for 2 minutes at room temperature. Caco-2 cells and bacteria were then coincubated for 30 minutes at 37°C, followed by 2 washes with warm MEM. Caco-2 cells were further incubated for the indicated times in the presence of conditioned or control medium supplemented with 15 μg/mL gentamicin. Cells were washed 3 times with warm PBS. A volume of 300 μL PBS containing 0.2% Triton X-100 was added to each well to lyse the Caco-2 cells. Cell lysates were diluted and plated on BHI agar plates to enumerate bacterial colony-forming units.
Mimic transfections
Mimics for C19MC miRNAs (miRIDIAN; Dharmacon, Lafayette, CO) as well as a nontargeting control miRNA mimic were obtained from Thermo-Fisher USA as previously described. U2OS cells were reverse transfected with miRNA mimics or miRNA mimic control (final concentration, 25 nmol/L for each miRNA mimic), using DharmaFECT-1 transfection reagent (Thermo-Fisher) according to the manufacturers’ instructions. Cells were assayed 48 hours posttransfection.
Statistics
Experiments were performed at least 3 times as indicated in Figures 2-6 . Data are presented as mean ± SD. Except where specified, Student t test was used to determine statistical significance for virus infections when 2 sets were compared, and 1-way analysis of variance, with Bonferroni correction for post hoc analysis of multiple comparisons, was used to determine statistical significance for reporter gene assays; P < .05 was considered significant.
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