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

View Article: PubMed Central - PubMed

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.

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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×.
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viruses-08-00241-f003: 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×.

Mentions: Since we observed a clear difference in stratification patterns between ALI and LLI cultures, we next measured the ability of cells to express cytokeratin, an epithelial cell specific protein, to study the differentiation of Vk2 cells under these distinct conditions. Since vaginal cells and their functions, including growth and differentiation, are profoundly affected by female sex hormones, we decided to compare cytokeratin expression in the presence of E2, P4, or MPA. The hormone concentrations used for these studies were based on the serum concentrations of E2 and P4 seen under physiological conditions (menstrual cycle E2: 10−11 M to 10−10 M and P4: 10−9 M to 10−8 M; pregnancy E2: 10−9 M and P4: 10−6 M) and serum levels of Depot medroxyprogesterone acetate (DMPA) observed following subcutaneous injection (1 ng/mL) and have been described before [26]. Exposing Vk2 cells to different hormones over 10 days did not affect cell viability (Figure 3A).


Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface
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×.
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Related In: Results  -  Collection

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viruses-08-00241-f003: 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×.
Mentions: Since we observed a clear difference in stratification patterns between ALI and LLI cultures, we next measured the ability of cells to express cytokeratin, an epithelial cell specific protein, to study the differentiation of Vk2 cells under these distinct conditions. Since vaginal cells and their functions, including growth and differentiation, are profoundly affected by female sex hormones, we decided to compare cytokeratin expression in the presence of E2, P4, or MPA. The hormone concentrations used for these studies were based on the serum concentrations of E2 and P4 seen under physiological conditions (menstrual cycle E2: 10−11 M to 10−10 M and P4: 10−9 M to 10−8 M; pregnancy E2: 10−9 M and P4: 10−6 M) and serum levels of Depot medroxyprogesterone acetate (DMPA) observed following subcutaneous injection (1 ng/mL) and have been described before [26]. Exposing Vk2 cells to different hormones over 10 days did not affect cell viability (Figure 3A).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus