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

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

The anti-inflammatory activity of curcumin protects the genital mucosal epithelial barrier from disruption and blocks replication of HIV-1 and HSV-2.

Inflammation is a known mechanism that facilitates HIV acquisition and the spread of infection. In this study, we evaluated whether curcumin, a potent and safe anti-inflammatory compound, could be used to abrogate inflammatory processes that facilitate HIV-1 acquisition in the female genital tract (FGT) and contribute to HIV amplification. Primary, human genital epithelial cells (GECs) were pretreated with curcumin and exposed to HIV-1 or HIV glycoprotein 120 (gp120), both of which have been shown to disrupt epithelial tight junction proteins, including ZO-1 and occludin. Pre-treatment with curcumin prevented disruption of the mucosal barrier by maintaining ZO-1 and occludin expression and maintained trans-epithelial electric resistance across the genital epithelium. Curcumin pre-treatment also abrogated the gp120-mediated upregulation of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6, which mediate barrier disruption, as well as the chemokines IL-8, RANTES and interferon gamma-induced protein-10 (IP-10), which are capable of recruiting HIV target cells to the FGT. GECs treated with curcumin and exposed to the sexually transmitted co-infecting microbes HSV-1, HSV-2 and Neisseria gonorrhoeae were unable to elicit innate inflammatory responses that indirectly induced activation of the HIV promoter and curcumin blocked Toll-like receptor (TLR)-mediated induction of HIV replication in chronically infected T-cells. Finally, curcumin treatment resulted in significantly decreased HIV-1 and HSV-2 replication in chronically infected T-cells and primary GECs, respectively. All together, our results suggest that the use of anti-inflammatory compounds such as curcumin may offer a viable alternative for the prevention and/or control of HIV replication in the FGT

Curcumin prevents tight junction disruption and breakdown of endometrial epithelial barrier integrity caused by HIV-1 gp120.

<p>Primary endometrial GECs were grown to confluency and pre-treated with 5μM curcumin or media as control for 1 hour and exposed to 10⁵ IVU of HIV-IIIB, recombinant gp120 at 0.1μg/mL or media (mock infection). At 24 hours post exposure, GECs were fixed and stained for ZO-1 (A) or occludin (B). Imaging was done on an inverted confocal laser-scanning microscope. Alternatively, GECs were exposed to media control (untreated), gp120 at 0.1μg/mL or recombinant TNF-α at 100 ng/mL (positive control) for 24 hours, after which TERs were measured and the percent of pre-treatment TER was calculated (C). Data shown represents the mean ± SEM of three separate experiments. A minimum of two replicates per experimental condition was included in every experiment. Data was analyzed by Kruskal-Wallis non-parametric analysis of variance with Dunn’s test to correct for multiple comparisons. **p<0.01, ***p<0.001. gp120: glycoprotein 120; HIV-1: human immunodeficiency virus-1; TER: trans-epithelial electrical resistance; TNF-α: tumor necrosis factor-α.</p

Curcumin can prevent co-infection mediated activation of the HIV-LTR promoter and induction of HIV replication in T-cells.

<p>Confluent primary endometrial GECs were pre-treated with 5 μM curcumin or primary cell media (untreated) for 1 hour after which the cells were exposed to 10⁴ PFU of HSV-1, HSV-2 or 10⁶ CFU of <i>N</i>. <i>gonorrhoeae</i> for two hours. The inoculum was removed and the cells were washed with PBS and subsequently replenished with fresh media. Supernatants were collected 24-hours post-exposure, inactivated to kill live virus or filter sterilized to remove bacterial cells, and subsequently incubated with 10⁶ 1G5 cells for 24 hours, after which the cells were lysed and luciferase activity was measured as a readout for HIV-LTR activation (A). To assess 1G5 cell viability following exposure to conditioned GEC media, 1G5 cells were exposed for 24 hours to RPMI control (Media), primary cell media from uninfected GECs (Uninfected) and conditioned primary cell media from GECs exposed to HSV-1, HSV-2 or <i>N</i>. <i>gonorrhoeae</i> for 24 hours. Following incubation, cells were collected and cell viability was calculated using the trypan exclusion assay. Results are reported as the percent of live cells in culture (B). 1x10⁵ chronically infected H9 T-cells were pre-treated with 50 μM of curcumin for 1 hour and subsequently exposed to poly I:C, LPS or flagellin for 24 hours. Supernatants were collected and IVUs were measured using the TZM-b1 assay (C). Data shown represents the mean ± SEM of three separate experiments. A minimum of two replicates per experimental condition were included in every experiment performed. Data was analyzed using a two-tailed Mann-Whitney test for non-parametric data. *p<0.05, **p<0.01, ***p<0.001. HIV-LTR: human immunodeficiency virus long-terminal repeats; RLU: relative light units; HSV: herpes simplex virus; NGO: <i>Neisseria gonorrhoeae</i>.; LPS: lipopolysaccharide.</p

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of Th1 and Th17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher Th17 and Th1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c+ DCs in the vagina were the predominant APC population responsible for priming these Th17 responses, and a potent source of IL-6 and IL-1β, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored Th17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient Th1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4+ T cell anti-viral immunity by priming vaginal DCs to induce Th17 responses through an IL-1-dependent pathway

Correction: Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

[This corrects the article DOI: 10.1371/journal.ppat.1005589.]

E2 pre-treatment enhances protection against genital HSV-2 challenge in intranasally immunized mice.

<p>WT (C57Bl/6) OVX mice treated with E2 or placebo pellets, were intranasally immunized 1 week and 3 weeks later with 1x10³ TK⁻ HSV-2, or 5μg HSV-2 gD + 30μg CpG, or 1x10⁴ pfu HI HSV-2 333 + 30μg CpG (n = 5–10 mice/hormone group for each vaccine formulation). Five weeks following the second immunization, all groups of mice were intravaginally challenged with 5x10³ pfu/mouse WT HSV-2 333. (A) Survival curves showing the percentage of mice that survived WT HSV-2 challenge in all vaccine formulations. Significance in difference in survival was calculated using the log-rank (Mantel-Cox) test (* p<0.05, ** p<0.01). (B) Pathology scores in these mice were graded on a 1–5 scale as described in the Materials and Methods section, and plotted. Data points superimposed on X-axis indicate mice without genital pathology, and the % indicates maximum number of mice that showed pathology. (C) Vaginal washes were collected for 5 days post-challenge, and HSV-2 viral shedding (bar indicates mean pfu/mL of shed virus) was calculated by conducting viral titrations with a vero-cell based assay. Dashed line indicates the lower detection limit of this assay, and data points on this line indicate undetectable viral shedding. The % indicates maximum number of mice that shed virus between days 1 to 5 post-challenge. Each symbol in B and C represents a single animal and data has been pooled from two separate experiments with similar results.</p

Cytokine production by CD4⁺ T cells in the vagina post intravaginal HSV-2 challenge.

<p>Cytokine production by CD4⁺ T cells in the vagina post intravaginal HSV-2 challenge.</p