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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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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.
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viruses-08-00241-f005: 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.

Mentions: To confirm the effect of P4 on the enhancement of HSV-2 infection, we measured the amount of virus shed by the Vk2 cells in ALI or LLI cultures, an indicator of virus replication, following each sex hormone treatment. Confluent Vk2 cultures exposed to sex hormones in ALI or LLI conditions were infected with wild-type HSV-2, and at 24 h post-infection, apical and basolateral cell culture supernatants were collected to measure viral shedding. Overall, Vk2 cells in the ALI culture showed higher viral shedding than in the LLI culture (Figure 5). In LLI, cells exposed to P4 and MPA showed significantly higher viral shedding than when exposed to E2 and the no hormone control (Figure 5A), while in ALI, P4 group showed significantly higher viral titers than all other treatment groups (p < 0.0001) (Figure 5B). Thus, the trend of the P4 group under ALI conditions displaying the highest infectivity and the E2 group displaying the lowest was consistent with the HSV-2-GFP infection results in ALI (Figure 4B). Overall there was consistency between the ALI and LLI cultures in that both showed significant increase in HSV-2 shedding in the P4 group compared to the control (no hormone) and E2. These results also suggest that E2 may play a protective role against HSV-2 infection.


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

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getmorefigures.php?uid=PMC5035955&req=5

viruses-08-00241-f005: 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.
Mentions: To confirm the effect of P4 on the enhancement of HSV-2 infection, we measured the amount of virus shed by the Vk2 cells in ALI or LLI cultures, an indicator of virus replication, following each sex hormone treatment. Confluent Vk2 cultures exposed to sex hormones in ALI or LLI conditions were infected with wild-type HSV-2, and at 24 h post-infection, apical and basolateral cell culture supernatants were collected to measure viral shedding. Overall, Vk2 cells in the ALI culture showed higher viral shedding than in the LLI culture (Figure 5). In LLI, cells exposed to P4 and MPA showed significantly higher viral shedding than when exposed to E2 and the no hormone control (Figure 5A), while in ALI, P4 group showed significantly higher viral titers than all other treatment groups (p < 0.0001) (Figure 5B). Thus, the trend of the P4 group under ALI conditions displaying the highest infectivity and the E2 group displaying the lowest was consistent with the HSV-2-GFP infection results in ALI (Figure 4B). Overall there was consistency between the ALI and LLI cultures in that both showed significant increase in HSV-2 shedding in the P4 group compared to the control (no hormone) and E2. These results also suggest that E2 may play a protective role against HSV-2 infection.

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