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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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Related in: MedlinePlus

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.
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viruses-08-00241-f002: 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.

Mentions: To determine whether the ALI conditions lead to epithelial cell cultures that physiologically simulate in vivo vaginal cells compared to the LLI conditions, we first examined the cell growth and morphology in these cultures through H&E staining and F-actin staining. Confluent Vk2 cells were fixed and stained after three and 10 days of culture. Vk2 cells in LLI culture grew as monolayers of simple squamous epithelium, while the cells in ALI culture grew and differentiated into multilayered, stratified squamous epithelium, which is analogous to the vaginal morphology in vivo (Figure 2). These results provided evidence that ALI culture conditions resemble more closely the vaginal cells in vivo and could potentially be a more relevant model to study the vaginal cell interactions in the lower FRT.


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

License
Show All Figures
getmorefigures.php?uid=PMC5035955&req=5

viruses-08-00241-f002: 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.
Mentions: To determine whether the ALI conditions lead to epithelial cell cultures that physiologically simulate in vivo vaginal cells compared to the LLI conditions, we first examined the cell growth and morphology in these cultures through H&E staining and F-actin staining. Confluent Vk2 cells were fixed and stained after three and 10 days of culture. Vk2 cells in LLI culture grew as monolayers of simple squamous epithelium, while the cells in ALI culture grew and differentiated into multilayered, stratified squamous epithelium, which is analogous to the vaginal morphology in vivo (Figure 2). These results provided evidence that ALI culture conditions resemble more closely the vaginal cells in vivo and could potentially be a more relevant model to study the vaginal cell interactions in the lower FRT.

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