Limits...
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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5035955&req=5

viruses-08-00241-f004: 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.

Mentions: Since the effect of hormones on epithelial cell differentiation appeared to be enhanced in ALI conditions, we next examined the effects of female sex hormones on susceptibility to HSV-2 infection. We infected Vk2 cells grown in ALI and LLI conditions with a GFP-labelled thymidine kinase HSV-2 virus (HSV-2-GFP) and used fluorescence microscopy to visualize the infection. The relative fluorescence of the HSV-2-GFP (green) and the nuclear stain (red) was measured using the ImageJ software and their ratio was calculated in each field, in order to estimate the viral infection per cell. Under ALI conditions, Vk2 cells grown and exposed to HSV-2-GFP in the presence of P4 showed a significantly higher infection per cell compared to the E2 group (Figure 4A). A similar trend of higher relative fluorescence was seen in the P4 group in LLI cultures as well, but did not reach statistical significance. Overall, the highest infections were visualized in fluorescence microscopy images of the P4 conditions in both ALI and LLI (Figure 4B). These results indicate that P4 may play a role in increasing the susceptibility of Vk2 to HSV-2, compared to E2 treated cultures.


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

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

viruses-08-00241-f004: 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.
Mentions: Since the effect of hormones on epithelial cell differentiation appeared to be enhanced in ALI conditions, we next examined the effects of female sex hormones on susceptibility to HSV-2 infection. We infected Vk2 cells grown in ALI and LLI conditions with a GFP-labelled thymidine kinase HSV-2 virus (HSV-2-GFP) and used fluorescence microscopy to visualize the infection. The relative fluorescence of the HSV-2-GFP (green) and the nuclear stain (red) was measured using the ImageJ software and their ratio was calculated in each field, in order to estimate the viral infection per cell. Under ALI conditions, Vk2 cells grown and exposed to HSV-2-GFP in the presence of P4 showed a significantly higher infection per cell compared to the E2 group (Figure 4A). A similar trend of higher relative fluorescence was seen in the P4 group in LLI cultures as well, but did not reach statistical significance. Overall, the highest infections were visualized in fluorescence microscopy images of the P4 conditions in both ALI and LLI (Figure 4B). These results indicate that P4 may play a role in increasing the susceptibility of Vk2 to HSV-2, compared to E2 treated cultures.

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