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BOR - Papers in Press, published online ahead of print September 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063933
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BIOLOGY OF REPRODUCTION 77, 1049–1059 (2007)
DOI: 10.1095/biolreprod.107.063933
© 2007 by the Society for the Study of Reproduction, Inc.

Susceptibility of Human Female Primary Genital Epithelial Cells to Herpes Simplex Virus, Type-2 and the Effect of TLR3 Ligand and Sex Hormones on Infection1

Erin M MacDonald , Alison Savoy , Amy Gillgrass , Sherie Fernandez , Marek Smieja , Kenneth L Rosenthal , Ali A Ashkar , and Charu Kaushic  2

Center For Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Michael G. DeGroote Center for Learning and Discovery, Hamilton, Ontario, Canada L8P 3Z5

ABSTRACT

Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.

female genital epithelium, HSV-2, innate immunity, sex hormones, sexually transmitted virus, susceptibility, TLR

INTRODUCTION

Sexually transmitted infections (STIs) are a cause of severe morbidity worldwide, and there are approximately 200–300 million new cases every year [1]. The mucosal surface in the genital tract is the primary route of infection of most sexually transmitted infections; more than 70% of human immunodeficiency virus (HIV)-1 transmission occurs across the genital epithelium [2]. Additionally, the rate of transmission is much greater from males to females than it is from females to males [3]. This difference in transmission, along with other biological factors, likely accounts for higher incidence of STIs in women. UNAIDS figures show that heterosexual transmission is driving the AIDS pandemic, and women account for nearly 50% of HIV-infected patients [4]. Despite these facts, the interaction of sexually transmitted pathogens with the female genital tract is poorly understood.

Herpes simplex virus, type 2, (HSV-2) is arguably the most common sexually transmitted virus. An estimated 20% of North American, 15% of European, and 50% of sub-Saharan African adults are infected with HSV-2, with a higher prevalence in women than men [5–7]. Genital herpes infection occurs when HSV-2 comes in contact with the mucosal epithelium or abraded skin and initiates infection. The exact mechanism by which HSV-2 enters the female genital epithelium is not known, though studies in cell lines indicate that the initial attachment is likely mediated through viral glycoproteins C and B interacting with cell surface heparan sulfate proteoglycans. The actual entry of the virus is usually receptor mediated and may involve binding of specific cellular receptors including Hve A, B, or C by viral glycoprotein D [8–10].

Most information on HSV pathogenesis comes from in vitro infection of either epithelial cell (EC) lines or keratinocytes by HSV-1 [11–15]. These studies have shown a wide variability in the ability of HSV to infect cells from the apical and basolateral surfaces, depending on the type of cell and experimental conditions. In vitro culture systems of primary ECs from the human genital tract have also been described previously (reviewed in [16]). Many of these studies have focused on characterization of EC morphology and functions from physiologically normal uterus or endometriosis patients [17–20]. Interactions of stromal-epithelial cells, the effect of steroids on EC polarity, growth factors, and proteins secreted by endometrial ECs have also been extensively examined in these culture systems [16, 21–23]. However, so far no studies have examined the infectivity of primary ECs from the female genital tract to HSV. The present study was undertaken to examine the susceptibility of primary human ECs from endometrial and cervical tissue to HSV-2 in this ex vivo culture system. We show that this culture system can provide valuable information to better understand viral pathogenesis as well as modifiable factors that can change susceptibility of genital ECs.

MATERIALS AND METHODS

Source of Tissues

Reproductive tract tissues were obtained from women aged 30–59 yr (mean age 42.93 ± 7.2) undergoing hysterectomy for benign gynecological reasons at Hamilton Health Sciences Hospital. Informed consent was received in accordance with the approval of the Hamilton Health Sciences Research Ethics Board. The most common reasons for surgery were uterine fibroids and heavy bleeding.

Epithelial and Stromal Cell Preparation

Endometrial and cervical tissues from hysterectomy patients were obtained within 1–3 h of surgery from the Clinical Pathology Laboratory at Hamilton Health Sciences Center. Coin-sized pieces were removed from tissues deemed by the pathologists to be free from malignancy and any other clinically observed disease. Tissue pieces were taken from the mucosal side of the endometrium and endocervix. Excess submucosal tissue was removed to get pieces consisting mostly of epithelium and stroma. Tissues were placed immediately in cold Hanks Balanced Salt Solution (HBSS) (Invitrogen, Burlington, ON, Canada). Isolation of ECs began as soon as tissue was bought into the laboratory. The EC isolation procedure was based on modification from previously described protocol [22]. Briefly, each tissue was minced into 1- to 2-mm pieces and digested in a solution of 20 ml HBBS and enzymes (1.5 mg/ml collagenase A [Roche, Montreal, QC, Canada], 3.45 mg/ml pancreatin [Sigma-Aldrich, Oakville, ON, Canada], 0.1 mg/ml hyaluronidase [Roche], and 0.2 mg/ml D-glucose [Sigma-Aldrich]) at 37°C for 1 h. Following digestion, the mixture was passed through 250-µm and 20-µm filters (Small Parts, Miami Lakes, FL).

Epithelial cells were harvested from the 20-µm filter. Cells were centrifuged and re-suspended in primary tissue culture media (Dulbecco modified Eagle medium [DMEM]:F12; Invitrogen) supplemented with 10 µM Hepes (Invitrogen), 2 µM L-glutamine (Invitrogen), 100 U/ml penicillin/streptomycin (Sigma-Aldrich), 2.5% Nu Serum culture supplement (Becton, Dickinson and Company, Franklin Lakes, NJ), and 2.5% Hyclone defined fetal bovine serum (FBS; Hyclone, Logan, UT). To remove stromal cells, the suspension was plated in a culture dish for 1 hour at 37°C with 5% CO2 to allow fibroblasts to adhere to the plastic. ECs were plated on Matrigel (Becton, Dickinson and Company)-coated BD Falcon 0.4-µm pore tissue culture inserts (Becton, Dickinson and Company) in plastic 24-well plates (Becton, Dickinson and Company) at an approximate density of 1–2 x 105 cells per insert in 300 µl of primary culture media, with 700 µl of primary culture media added to the basolateral side of the insert. Primary culture media was replaced after 24 h of culture and subsequently changed every 48 h. An Epithelial Voltohmmeter (EVOM) (World Precision Instruments, Sarasota, FL) with the STX2 chopstick electrode was used to determine the transepithelial electrical resistance (TER) of the epithelial monolayers grown in tissue culture inserts. TERs were used to indicate epithelial growth and the formation of tight junctions. Polarized confluent epithelial monolayers were used for all experiments.

Stromal cells were isolated from the same tissue as the ECs. After digestion and filtration of the tissue, the filtrate from the 20-µm filter was collected in a 50-ml Falcon tube and centrifuged at 1800 rpm at 20°C. Cells were resuspended in 3 ml of PBS, underlayed with 3 ml of 100% Ficoll (Amersham Biosciences, Baie d'Urfe, QC, Canada) and centrifuged at 1200 rpm at 20°C. The stromal cells at and above the interface were collected and washed once in primary culture media. Stromal cells were plated into plastic 24-well plates (Becton, Dickinson and Company) at a concentration of approximately 4–5 x 105 cells per well in 300 µl of primary culture media. Primary culture media was replaced after 24 h of culture and subsequently changed every 48 h.

For epithelial-stromal cell co-cultures, stromal cells were grown on plastic 24-well plates (Becton, Dickinson and Company). Confluent ECs in tissue culture inserts were added to wells containing stromal cells from the same patient.

Hematoxylin Staining of Confluent Endometrial and Cervical Epithelial Monolayers

To visualize epithelial monolayers, tissue culture inserts were stained with Harris hematoxylin (Sigma-Aldrich). Inserts were washed with PBS for 10 min, fixed with 100% ethanol for 10 min, and washed with PBS for 10 min. Harris hematoxylin was added for 1 min and washed extensively with tap water. Inserts were dried and membranes were cut out from the plastic wells. Membranes were mounted onto glass slides with glass cover slips in Permount Mounting Media (Sigma-Aldrich).

Flow Cytometric Analysis

Single ECs for flow analysis were obtained by incubating EC culture inserts with 1% trypsin-EDTA (Sigma-Aldrich) at 37°C for 5 min to generate a single cell suspension. The cells were then fixed and permeabilized using a commercial kit (BD Biosciences Canada, Mississauga, ON, Canada), prior to incubation with fluorescent antibodies or control antibodies. Murine monoclonal antibodies used for flow cytometry were conjugated with fluorescein isothiocyanate (FITC). Antibodies to ECs included a cytokeratin antibody that binds to an EC intracellular filament protein and a BerEp4 antibody that binds to an extracellular EC protein (Dako Cytomation, Mississauga, ON, Canada). Leukocyte antibody anti-CD45 (BD Biosciences Canada) was also used. Intracellular staining was performed with the Dako Intrastain Kit (Dako Cytomation) according to the manufacturer's instructions. Samples were analyzed on the BD FACS Scan, acquiring 20 000 events. Raw data was analyzed with Verity WinList 2.0 (Verity Software House, Topsham, ME) and WinMDI 2.8 (Joseph Trotter, Scripps, San Diego, CA) analysis software.

Immunohistochemical Staining

Monoclonal antibodies against human cytokeratin (EC marker) and vimentin (fibroblast marker; Dako Cytomation) were used for characterizing genital cell cultures by immunohistochemistry. IgG1k was used as an isotype control. The secondary antibody was the goat anti-mouse Envision (Dako Cytomation) system, and liquid substrate-chromogen DAB (Dako Cytomation) was used for visualization. Membranes were cut from the inserts, mounted on glass slides, and sealed with Crystal Mount aqueous mounting media prior to microscopic examination.

Apical and Basolateral Infection of EC Cultures with HSV-2

HSV-2 strain 333 was used for all EC culture insert infections. Confluent monolayers of endometrial and cervical ECs with TERs above 700 ohms were used for all infections. Cells were infected via the apical surface with a standard inoculation dose of 104 plaque-forming units (pfu) of HSV-2 in a volume of 100 µl. The virus was incubated with cells for 2 h at 37°C in 5% C02 and washed five times with PBS. The final wash was collected for viral titering to ensure there was no HSV-2 left after washing. Three hundred microliters of fresh primary culture media was layered in the apical and basolateral chambers for the duration of the infection. Apical and basolateral supernatants were collected 24 h postinfection and stored at –70°C for viral titering using a standard plaque-forming Vero cell assay, as described before [24]. Cells were infected via the basolateral surface with a dose of 1–5 x 106 pfu of HSV-2 in a volume of 300 µl. Virus was incubated with cells for 2 h at 37°C in 5% C02, rocked every 15 min, and washed five times basolaterally with PBS. The last wash was collected for viral titering to ensure there was no HSV-2 left after washing. To establish viral infection kinetics for both apical and basolateral infections, supernatants were collected 4, 12, 18, 24, and 48 h postinfection and stored at –70°C for viral titering.

For HSV-2-GFP infection (kindly donated by Dr. Jeff Viera, Fred Hutchinson Cancer Research Center Program in Infectious Diseases, Seattle, WA), confluent endometrial and cervical ECs were grown on glass chamber culture slides coated with Matrigel. Confluent cultures were infected with 104 and 105 pfu of HSV-2-GFP for 24 h. After infection, cells were washed with PBS and fixed in 4% paraformaldehyde. Slides were mounted with Fluorescent Mounting Media (Sigma-Aldrich) and kept at 4°C in the dark until observed with a scanning laser confocal microscope (LSM-510l; Carl Zeiss, Thornwood, NY). Slides were subjected to excitation with the FITC narrow band region to observe florescence of infected cells.

Infection of EC Cultures in the Presence of Sex Hormones and Following Antiviral Treatment

Experiments to test the effect of hormones and antiviral formulations on HSV-2 infection of EC culture inserts were carried out according to the standard protocol, with the following modifications. In hormone experiments, prior to infection, cultures were incubated for 24 h in hormone-stripped media (Phenol red-free DMEM:F12 [Invitrogen] supplemented with 10 µM HEPES, 2 µM L-glutamine, 100 U/ml penicillin/streptomycin, 12.5 µg of fungizone, and 5% charcoal/dextran-treated FBS) to remove the effects of any hormones present in the primary culture media, and then incubated for an additional 24 h with hormone-stripped media as a control or with hormone-stripped media containing 10–9 M 17β-estradiol or 10–6 M progesterone (Sigma-Aldrich). The virus was diluted in either the hormone-stripped media or stripped media containing 10–9 M 17β-estradiol or 10–6 M progesterone, and after 2 h of infection, cells were washed and provided with fresh hormone-stripped media with or without hormones. In experiments testing the effect of antivirals, endometrial and cervical monolayers were pretreated apically and basolaterally with 300 µl of 10 µg/ml poly (I:C) (Sigma-Aldrich) for 20–24 h or left untreated as control. Following the 2-h infection, cells were washed, and 300 µl of 10 µg/ml of poly (I:C) or primary culture media was added to the apical and basolateral compartments. Acyclovir (ACV) (Sigma-Aldrich) was used to treat endometrial and cervical EC monolayers after infection with HSV-2. EC cultures were treated with either 200 µM ACV or left untreated as controls.

Statistical Analysis of Data

GraphPad Prism 3.02 (GraphPad Software, San Diego, CA) was used for statistical analysis and graphic representation. All viral quantitation was log transformed to improve normality for parametric statistical analysis. Time-kinetic experiments were analyzed with one-way analysis of variance (ANOVA), and pairs of individual time points were compared using the two-sample Student t-test. Poly (I:C) and hormone experiments were analyzed with Student paired t-tests. All t-tests were performed two-tailed, and significance defined at an alpha value of 0.05.

RESULTS

Characterization of Endometrial and Cervical Epithelial and Stromal Cell Cultures

Primary human endometrial and cervical EC cultures were examined for growth to confluence and for purity. The TER measurement was used to monitor establishment of tight junctions as polarized EC cultures reached confluence. The TER values of typical endometrial EC monolayers increased from 150 ohms at the initiation of cultures to 800–1200 ohms after 5–7 days in culture (Fig. 1A). Cervical EC monolayers in general had higher TER values, up to 4500 ohms after approximately 7–8 days in culture (Fig. 1A). Endometrial and cervical EC monolayers were viable for approximately 20 days in culture, after which TERs fell below 700 ohms. The presence of high TER values indicated that the cultures were enriched for columnar ECs that formed tight junctions. EC cultures were found to be confluent by hematoxylin staining (Fig. 1B).


Figure 01
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  		FIG. 1 Growth of endometrial and cervical epithelial monolayers. A) Average TERs from Day 2 to 20 on endometrial and cervical EC cultures. Graph is a representation based on the TERs of one patient's endometrial and cervical EC culture. Control TERs were measured from cell-culture inserts coated with Matrigel, but without any cells. Note that in this patient, the cervical TERs started to increase at Day 8 of culture. B) Following 7–10 days in culture, endometrial and EC monolayers were hematoxylin stained and mounted on glass slides. Magnifications x100 and x200 (insets).

To confirm the purity of the ECs, cultured monolayers were disaggregated and double stained for the intra- and extracellular epithelial-specific markers cytokeratin and BerEp4 and examined by flow cytometry. Endometrial cultures were 98.5% positive, and cervical monolayers were 94.5% positive by double staining (Fig. 2A). No staining above background levels was observed for CD45, a marker for hematopoeitic cells, in either endometrial or cervical EC culture, indicating absence of any contamination by immune cells (Fig. 1B). Immunohistochemistry was also performed on confluent monolayers, using the EC marker cytokeratin and the stromal cell marker vimentin. All EC cultures stained positive for cytokeratin, with minimal to no staining visible for vimentin (Fig. 2C).


Figure 02
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  		FIG. 2 Endometrial and cervical epithelial cell culture purity. Endometrial and cervical monolayers were disaggregated after 7 days in culture and (A) double stained with cytokeratin and BerEp4-FITC or (B) single stained with CD45-FITC. C) Confluent endometrial and cervical monolayers were stained with cytokeratin or vimentin or isotype control. D) Stromal cultures were stained with cytokeratin or vimentin or isotype control. Magnifications are indicated in each photomicrograph.

Primary endometrial and cervical stromal cell cultures were also characterized for viability and purity. Both types of stromal cell cultures were examined with trypan blue exclusion and were found to completely exclude the dye for up to 20 days, indicating prolonged viability (data not shown). Stromal cell cultures from the endometrium and cervix were analyzed for purity by immunohistochemical staining for vimentin, a fibroblast cell marker, and cytokeratin, an epithelial marker. Stromal cultures consistently stained positively for vimentin with very little cytokeratin staining, indicating little, if any, EC contamination (Fig. 2D).

HSV-2-GFP Infection of Endometrial and Cervical EC Cultures

To determine the susceptibility of endometrial and cervical ECs grown ex vivo, confluent EC monolayers were inoculated apically with HSV-2-GFP to visualize infection. A dose curve was performed on EC cultures from a minimum of three patients to examine the optimal inoculation dose of HSV-2 for apical infections. Viral shedding increased with increasing inoculation doses from 103–106 pfu of HSV-2 (data not shown). At 24 h postinfection, both endometrial and cervical EC had active infection, as indicated by GFP fluorescence seen inside the infected cells (Fig. 3). It was determined that 104 pfu was the optimal dose because infection with 105 pfu or higher appeared to saturate the infection of the monolayers with no further increase in viral shedding (data not shown).


Figure 03
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  		FIG. 3 Infection of primary cultures of endometrial and cervical ECs. Confluent endometrial and cervical ECs cultured on Matrigel-coated glass slides were infected with 105 pfu of HSV-2 GFP for 24 h. Cultures were fixed and mounted with fluorescent mounting media and viewed with a Carl Zeiss Scanning Confocal Microscope LSM 5–10 on the FITC narrow-band wave length. Magnification x630.

Reproducibility of HSV-2 Infection in Primary Cultures

Increased viral shedding after 18 h from infected endometrial and cervical EC cultures was a consistent observation between tissue samples. However, there was some variability in the absolute amount of HSV-2 produced by each patient culture. To determine if infection was reproducible within EC cultures established from the same patient, multiple inserts of endometrial and cervical EC cultures from separate patients were infected with the same dose (104 pfu) of HSV-2. Replicate culture inserts were terminated at 24 or 48 h postinfection. Viral shedding from replicate cultures of both endometrial and cervical ECs from individual patient cultures was highly reproducible at both 24 h postinfection (Fig. 4) and 48 h (data not shown). Basolateral viral shedding from endometrial EC cultures was only detected 48 h postinfection (data not shown). Cervical EC cultures showed basolateral shedding 24 h postinfection in two of four patient cultures (Fig. 4); all four patient cultures showed basolateral shedding at 48 h postinfection in cervical cultures (data not shown).


Figure 04
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  		FIG. 4 Consistency of HSV-2 viral shedding by infected endometrial and cervical epithelial monolayers. Confluent monolayers of endometrial and cervical ECs were infected with 104 pfu HSV-2 strain 333 for 24 h. Three replicates from each patient at each time point were measured. Endometrial and cervical patient cultures, n = 4.

Apical Versus Basolateral Infection of EC Cultures

Previous reports on infection of epidermal and EC lines and cultures have reported different tropism of HSV-1 virus for apical and basolateral infection. We therefore examined whether HSV-2 exhibited preferential infection when inoculated apically versus basolaterally, in our confluent primary genital EC cultures. Following inoculation, kinetics of viral shedding was followed for 48 h. Endometrial or cervical EC grown to confluence on culture-well inserts were infected apically with 104 pfu of HSV-2 strain 333. For basolateral infection, initial experiments showed viral shedding comparable to levels seen with apical infection, when EC cultures were infected with 106 pfu basolaterally. Therefore, this was used as the inoculation dose for all basolateral infections. HSV-2 shedding was determined after 4, 12, 18, 24, and 48 h postinfection.

In endometrial cultures, following apical infection, the virus was first detected 18 h postinfection in apical culture supernatants and continued to accumulate over the 48 h that the infection was monitored (P < 0.01; Fig. 5B). However, very little virus was detected in the basolateral compartment until 48 h (Fig. 5C). Following basolateral infection, HSV-2 was seen in appreciable amounts in the basolateral supernatants, but there was no accumulation of virus over the course of infection (Fig. 5E). Although viral shedding started slowly in the apical supernatants following basolateral infection, preferential accumulation of virus was observed in the apical compartment over the course of infection (P < 0.01; Fig. 5D).


Figure 05
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  		FIG. 5 Apical versus basolateral infection of endometrial EC cultures. Confluent monolayers of endometrial were infected with 104 pfu of HSV-2 strain 333 apically or basolaterally with 106 pfu for 2 h, and apical and basolateral supernatants were collected at 12, 18, 24, and 48 h. Viral shedding in the supernatants was measured by Vero cell assay (Materials and Methods). A) Comparison of plaques seen in epithelial monolayers at various time points following apical versus basolateral infection. B, C) Viral titers after apical infection. Preferential accumulation of virus in the apical compartment. *, P < 0.001 (ANOVA), n = 5. D, E) Viral titers after basolateral infection. Increase in apical viral shedding from 18 to 48 h. *, P < 0.001 (ANOVA). More consistent shedding was seen in the basolateral compartment, though there was no preferential accumulation. n = 2–8 at different time points.

When monolayers of endometrial epithelial layers were stained at various time points, infection seemed to progress faster, as seen by plaques formed in the monolayer, following apical infection compared to basolateral infection, despite the higher inoculation dose (Fig. 5A).

Similar kinetics of infection were observed in cervical epithelial monolayers (Fig. 6). Again a preferential accumulation of HSV-2 was observed in apical supernantants over the course of infection following both apical and basolateral infection (P < 0.01; Fig. 6, A and C).


Figure 06
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  		FIG. 6 Apical versus basolateral infection of cervical EC cultures. Confluent monolayers of cervical ECs were infected with 104 pfu of HSV-2 strain 333 apically or basolaterally for 2 h, and apical and basolateral supernatants were collected at 12, 18, 24, and 48 h. Viral shedding in the supernatants was measured by Vero cell assay (Materials and Methods). A, B) Viral titers after apical infection. Cervical apical viral shedding increased from 18 to 24 h of infection (P < 0.01) and 24 to 48 h of infection. *, P < 0.05 (ANOVA), n = 4. C, D). Viral titers following basolateral infection. Significant increase in viral accumulation between 18 and 48 h. *, P < 0.01 (ANOVA), n = 4–7 at different time points.

Effect of Antiviral Treatments on HSV-2 Infection of EC Monolayers

Poly (I:C), a TLR3 ligand, has been shown to provide protection against HSV-2 infection in an in vivo mouse model of HSV-2 infection [25]. However, it is not clear from these studies whether poly (I:C) acts directly on ECs. The effectiveness of poly (I:C) to induce an antiviral effect against HSV-2 was tested in our human genital EC cultures. We used ACV treatment as a reference point to compare the effectiveness of poly (I:C) to inhibit the HSV-2 infection in cultured EC; ACV is an established clinical treatment for HSV-2. Endometrial EC culture inserts were treated with poly (I:C), ACV, or left untreated as controls. EC cultures were then infected apically, and viral shedding was measured after 24 h. Untreated control cultures shed a mean of 105 pfu/ml of HSV-2 into apical supernatants, but EC cultures treated poly (I:C) shed 1000-fold fewer HSV-2 (Fig. 7A). The ACV control indicated complete inhibition of virus production from EC cultures (fewer than 10 pfu/ml). Similar inhibition of HSV-2 replication was seen in cervical EC cultures (Fig. 7A). Virus shedding to the basolateral supernatants was not significant under any condition for EC cultures from this patient (Fig. 7A).


Figure 07
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  		FIG. 7 Effect of antiviral treatment on endometrial and cervical EC susceptibility to HSV-2 infection. A) Confluent monolayers of endometrial and cervical ECs were treated with poly (I:C) for 20 h prior to infection with 104 pfu/insert of HSV-2 for 24 h. Control inserts remained untreated prior to infection. After infection, previously untreated wells were treated with 200 µM ACV, and poly (I:C)-treated wells were treated with an additional 10 µg/ml of poly (I:C). Data points represent the mean production of HSV-2 by replicate wells of each patient culture treatment group. B) Confluent monolayers of endometrial and cervical ECs from different patients were treated with poly (I:C) for 24 h prior to infection with 104 pfu of HSV-2 strain 333 for 24 h. Each point represents the single-patient mean of three replicate cultures. n = 7 for endometrium, and n = 2 for cervical samples. *, P < 0.005 (paired t-test) for apical titers.

We then compared decreased HSV-2 shedding in response to poly (I:C) among EC cultures among different patient tissues. Endometrial EC cultures treated with 10 µg/ml poly (I:C) showed a decrease in apical viral shedding from a mean of 105 pfu/ml of HSV-2 to 103 pfu/ml of HSV-2 (n = 7; Fig. 7B). Similar results were obtained for cervical EC cultures (n = 2). In both cases the decline in HSV-2 shedding after treatment with poly (I:C) was significant (P < 0.05). These experiments confirmed the effectiveness of poly (I:C) to induce antiviral effects against HSV-2 infection directly on genital ECs and also showed the usefulness of the EC culture system for testing other antiviral agents.

Effect of Reproductive Hormones on HSV-2 Infection

Reproductive hormones are known to affect susceptibility to sexually transmitted viruses. In order to investigate how the sex hormones estradiol (E2) and progesterone (P4) may alter HSV-2 infection of human endometrial and cervical EC, we grew epithelial and stromal cell co-cultures. It is known that stromal cells are important for EC response to hormones [16, 23, 26]. E2 treatment, prior to HSV infection of endometrial EC-stromal co-cultures, resulted in a modest but significant increase in apical HSV-2 shedding after infection, compared with untreated controls (n = 5, P < 0.01; Fig. 8A). P4-treated endometrial co-cultures had a significant decrease (average 10-fold decrease) in HSV-2 viral shedding after infection (n = 5, P < 0.03; Fig. 8A). E2-treated cervical co-cultures showed variable results following infection with 104 pfu HSV-2 strain 333 (Fig. 8B). Four out of five patients showed decreased apical HSV-2 viral shedding following treatment. One patient culture showed an increase in HSV-2 shedding. No clear trends in viral shedding from cervical co-cultures were observed following P4 treatment (Fig. 8B).


Figure 08
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  		FIG. 8 Effect of 17β-estradiol and progesterone treatment on HSV-2 infection of endometrial and cervical epithelial monolayers. Confluent endometrial and cervical epithelial-stromal cell co-cultures were treated with media containing 10–9 M 17β-estradiol or 10–6 M progesterone or with hormone-free media as control for 24 h prior to infection with 104 pfu HSV-2 strain 333. Each data point is the mean of three replicate wells from individual patients. Each line represents the change in mean HSV-2 titers of controls compared to hormone-treated inserts from the same patient cultures. A) 10–9 M 17β-estradiol treatment (top row) of endometrial ECs increases HSV-2 production compared to hormone-free controls. *, P < 0.01 (paired t-test), n = 5. 10–6 M progesterone treatment (bottom row) decrease HSV-2 production compared to hormone-free controls. *, P < 0.03 (paired t-test), n = 5. B) 10–9 M 17β-estradiol treatment (top row) of cervical ECs compared to controls; P < 0.2446 (paired t-test), n = 5. 10–6 M progesterone treatment (bottom row) compared to controls, P < 0.2751 (paired t-test), n = 5.

DISCUSSION

This study shows that primary ECs isolated from endometrial and cervical tissue and grown into confluent, polarized monolayers in culture are susceptible to infection with HSV-2. Both endometrial and cervical cells in our cultures were predominantly columnar epithelial that formed tight junctions. This was evident from the high TERs seen in the cultures, indicating formation of polarized monolayers. We also demonstrated the purity of the cultures by immunohistochemistry and flow-cytometry staining techniques, to ensure the epithelial cultures were free from any significant contamination with any other cell type. Inoculation of the cultures with HSV-2-GFP clearly indicated that the virus was able to enter and replicate in the cells. The kinetics of the infection were very similar to those seen in other cell lines and in vivo, with significant viral shedding seen 18 h postinfection [24, 27–29]. Following both apical and basolateral infection, HSV-2 showed preferential shedding from the apical side of both endometrial and cervical ECs. The virus was rarely detected in the basolateral supernatants before 48 h of infection when infection was initiated apically. Viral shedding in the basolateral compartment was frequently seen 48 h postinfection and was most likely due to the cytopathic effect of HSV-2 on ECs, leading to loss of tight junctions and subsequent leaks in the monolayer. Following basolateral infection, there was a small but significant amount of HSV-2 present in the basolateral compartment that did not increase over time.

To the best of our knowledge, this is the first demonstration that genital ECs from women can be infected by HSV-2 ex vivo. Though this is not unexpected, the ability to demonstrate this in an ex vivo system allows us to examine the pathogenesis of genital ECs with direct clinical implications. Recently, we reported a murine primary genital culture system to examine HSV-2 infection [30]. Using this culture system, we found that ECs from the upper and lower reproductive tract of mice have different cytokine secretion profiles and respond differentially to HSV-2 infection, and that HSV-2 may be able to suppress epithelial cytokine secretion. Although in the present studies the infection kinetics were very similar in endometrial and cervical cultures, ongoing studies are examining whether there are differential cytokine secretion patterns following HSV-2 infection in human genital tissues.

One of the novel findings in our study was the observation that infection could be initiated in both endometrial and cervical cells following HSV-2 exposure on both apical and basolateral surfaces. However, 100 times more virus was required to infect basolaterally to achieve viral shedding to a similar extent as that seen following apical infection. Other studies have reported similar results whereby the presence of the membrane and filter in the transwell system may contribute to the decrease in the efficiency of infection [15]. The most striking observation in our study was that regardless of whether infection was initiated on the apical or basolateral side, HSV-2 appeared to be preferentially shed and accumulated within the apical compartment of epithelial cultures. Directional virus entry and release may have important implication in the spread of infection and transmission [31, 32]. In the context of the female genital epithelium, preferential apical release would indicate that HSV-2 infection can spread apically across the mucosal surface and infect other ECs without having to pass through microlesions [28]. In addition, preferential apical release could also increase the ability of the virus to be transmitted sexually.

Clinically, HSV-2 is primarily associated with infection of the vaginal epithelium in women. Although uncommon, intrauterine infection by HSV-2 has been reported in pregnant women [33]. Primary infection or re-activation of HSV-2 during pregnancy may lead to intrauterine transmission of HSV-2 to the fetus. The consequences of HSV-2 infection are quite severe for the fetus and neonate [34]. Such cases, though rare, lead to long-term neurological, skin, and liver sequelae and neonatal death. The ex vivo model described in this study provides a clinically relevant model to understand pathogenesis of HSV-2 in primary human genital cells and has direct implications in understanding the upper reproductive tract infection by HSV-2.

Previous studies have shown that the TLR3 ligand, synthetic dsRNA poly (I:C), has antiviral properties [35, 36]. In fact, there are clinical reports from two decades ago describing poly (I:C) treatment of neonatal HSV-2 infections [37]. More recently, poly (I:C) administered before challenge in a mouse model of HSV-2 infection was found to significantly decrease viral shedding compared to control mice [25]. These results suggest that poly (I:C) could be used to prevent HSV-2 infections or used to treat HSV-2 infections in the genital tract. In the present study, ECs were exposed to poly (I:C) prior to as well as during infection with HSV-2, to ensure antiviral protection was maintained. The decrease in HSV-2 shedding seen here was significant, but less than that seen following treatment with ACV, which completely inhibits viral replication and is a common treatment for genital herpes infection [38]. In subsequent studies, poly (I:C) exposure during infection was not found to be necessary; single pretreatment with poly (I:C) at a higher dose (25 µg/ml) 24 h prior to HSV-2 exposure was sufficient for complete protection (A. Nazli and C. Kaushic, unpublished results). Treatment of endometrial and cervical epithelial monolayers with poly (I:C) caused a significant decrease in viral shedding compared to control untreated wells. Poly (I:C) is a potent inducer of the antiviral factors interferon (IFN)-β, β defensins and IFN-β-stimulated genes in its target cells, which could be the mechanism by which it is acting to decrease viral shedding by the cultured ECs [36, 39, 40]. Our results clearly show that poly (I:C) can directly act on human genital ECs and result in a functional and potent antiviral effect. Our ongoing studies examining the mechanism of this antiviral effect in genital ECs show that protection is mediated by Type I IFN and NO (A. Nazli and C. Kaushic, unpublished results). The ability to induce a potent antiviral state following pretreatment of ECs raises the possibility of potential application for poly (I:C) for prophylactic treatment and microbicide formulations.

It is well established that women are more susceptible to STIs than men and that the cyclic changes in sex hormone levels in women affect their susceptibility to STIs [41–43]. Despite abundant clinical and epidemiologic data, there is no direct evidence that susceptibility of genital cells to sexually transmitted viruses is altered by reproductive hormones. Given these facts, our primary tissue culture system was evaluated for its ability to detect any changes in HSV-2 shedding in response to hormone treatment. Treatment of the cultures with the hormones estradiol and progesterone prior to HSV-2 infection resulted in small but significantly altered HSV-2 shedding. The ECs from both the endometrium and cervix express both estradiol and progesterone receptors (C. Kaushic et al., unpublished results). Treatment of endometrial ECs with 10–6 M of progesterone prior to infection with HSV-2 significantly decreased viral shedding. The concentration of progesterone used for these experiments was equivalent to the levels of progesterone from the late proliferative stage to the secretory phase of the menstrual cycle. It is an interesting possibility that there may be an evolutionary mechanism in the female genital tract whereby an increase in progesterone offers protection from invading pathogens without damage to the newly developing fetus. When endometrial ECs were treated with 10–9 M 17β-Estradiol and then infected, they were found to shed significantly more virus than untreated controls. These concentrations of estradiol are equivalent to those observed during the early follicular phase, which occurs just after menstruation has stopped early in the cycle and corresponds to endometrial proliferation. There may be other antiviral immune mechanisms in place that compensate for increased epithelial susceptibility under the influence of estradiol.

Several studies lend support to the results from hormone treatment experiments observed in our system. A study conducted with swine EC infection with Chlamydia trachomatis similarly found that progesterone treatment prevented ECs from becoming infected with the bacteria, whereas estradiol-treated cells were more susceptible to infection [44]. The swine reproductive cycle and EC physiology is similar to that of humans. Clinical prospective studies have also found similar results, though they are more controversial. Studies on HIV viral shedding have shown a decrease in the presence of HIV in cervical mucus when serum progesterone is high and an increase in HIV shedding in cervical mucus when serum estradiol levels are high [45].

In conclusion, this study provides direct evidence that HSV-2 can infect human female endometrial and cervical ECs apically and basolaterally and results in accumulation of virus on the apical side of the ECs. Further, our results show that TLR3 ligand poly (I:C) acts directly on human female genital ECs, resulting in antiviral effects. This implies that innate immune activators such as poly (I:C) can be successfully exploited for prophylactic microbicidal formulations. Our results also confirm clinical observations that show that reproductive hormones may directly affect STIs. This ex vivo primary epithelial culture system will be a very useful and relevant tool in understanding the mechanism of pathogenesis of sexually transmitted viruses, including HSV-2 and HIV-1. It will also be useful in developing and testing potential antiviral formulations.

ACKNOWLEDGMENTS

The authors acknowledge Jeff Viera (University of Washington) for kindly donating HSV-2-GFP and John Fahey (Dartmouth Medical School) for useful advice in standardizing the epithelial culture system. The authors thank Jen Newton for flow analysis and Mary Louis Beecroft for taking patient consents. We are also grateful to Fiona Smaill for useful suggestions and discussions regarding this work. We thank Denis Snider for critical reading and comments on this manuscript.

FOOTNOTES

1Supported by grants from the Canadian Institutes of Health Research (CIHR), Ontario HIV Treatment Network (OHTN), and Canadian Foundation for AIDS Research (CANFAR) to C.K. C.K. was supported by a Scholarship Award from OHTN and a New Investigator's Award from CIHR. E.M.M. and S.F. were supported by fellowships from OHTN. Back

Correspondence: 2Charu Kaushic, Department of Pathology, Center for Gene Therapeutics, MDCL 4014, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8P 3Z5. FAX: 905 522 6750; e-mail: kaushic{at}mcmaster.ca

Received: 5 July 2007.

First decision: 8 August 2007.

Accepted: 31 August 2007.

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