Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface

Abstract

The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.

Keywords: HSV-2; Vk2/E6E7 cells; air-liquid interface; estradiol; female genital tract; female sex hormones; herpes simplex virus type 2; medroxyprogesterone acetate; progesterone; vaginal cells.

Figure 1

Culture design comparison. (A) liquid-liquid interface (LLI); (B) air-liquid interface (ALI).

Figure 2

Hematoxylin and eosin (H&E) staining (A) and filamentous F-actin staining (B) comparison between the LLI and the ALI interface of Vk2 cells after 10 days of culture. Vk2 cells in ALI culture grew into multilayered epithelium compared to the monolayers seen in LLI. All samples were imaged on Leica HC DMR microscope under a 40× objective (Leica Microsystems). A minimum of three replicates per experimental condition was included. Representative images are shown.

Figure 3

Cytokeratin staining comparison between the LLI and ALI of Vk2 cells after treatment with female sex hormones and medroxyprogesterone acetate (MPA). (A) Vk2 cells grown to confluence for 10 days were exposed to no hormone (NH), estradiol (E2; 10⁻⁹ M), progesterone (P4; 10⁻⁷ M), and medroxyprogesterone acetate (MPA; 10⁻⁹ M). Cell viability was measured using the trypan blue assay and expressed as percent of live cells in culture; (B) Pancytokeratin staining, green fluorescence, was done using mouse anti-human monoclonal anti-pan cytokeratin antibody. Propidium iodide was used for nuclear counter-staining, red fluorescence. All samples were imaged on an inverted confocal laser-scanning microscope. Representative images are shown. Images were taken in the X, Y plane; (C) Relative fluorescence of cytokeratin (green) to nuclear stain (red) was measured to quantify the amount of cytokeratin present per cell. Data shown represent mean ± standard error of the mean (SEM) of three separate cultures. * p < 0.05. Three replicates per experimental condition were included for each experiment. Magnification 20×.

Figure 4

Effect of female sex hormones and MPA on HSV-2-GFP infection in Vk2 cells grown in ALI and LLI. Vk2 cells grown for 10 days to confluence in the presence or absence of female sex hormones were exposed to HSV-2-GFP at 10⁴ plaque-forming units (PFU) for two hours in both ALI and LLI. Nuclei were counter-stained with propidium iodide. (A) Images were taken to compare green fluorescence visually; (B) Relative fluorescence of HSV-2/GFP (green) to nuclear stain (red) was measured to quantify the amount of HSV-2 infection per cell. Data shown represented mean ± SEM of three separate cultures. ** p < 0.01. All samples were imaged on an EVOS™ FL digital inverted fluorescence microscope with a 20× objective. Images presented are representative of one of three images acquired from the experiments.

Figure 5

Difference in HSV-2 virus shedding by Vk2 cells in ALI and LLI conditions, exposed to different female sex hormones and MPA. To measure the viral shedding in Vk2 cell supernatants, Vk2 cells were exposed to 10⁴ PFU of wild-type HSV-2 for 2 h in both LLI (A) and ALI (B). Apical supernatants were collected and shed virus was measured by plaque assay on Vero cells (PFU/mL). Data were pooled from three separate experiments and are shown as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.