Novel Role for Interleukin-17 in Enhancing Type 1 Helper T Cell Immunity in the Female Genital Tract following Mucosal Herpes Simplex Virus 2 Vaccination.

It is well established that interferon gamma (IFN-γ) production by CD4+ T cells is critical for antiviral immunity against herpes simplex virus 2 (HSV-2) genital infection. However, the role of interleukin-17A (IL-17A) production by CD4+ T cells in HSV-2 antiviral immunity is yet to be elucidated. Here we demonstrate that IL-17A plays an important role in enhancing antiviral T helper type 1 (Th1) responses in the female genital tract (FGT) and is essential for effective protection conferred by HSV-2 vaccination. While IL-17A did not play a critical role during primary genital HSV-2 infection, seen by lack of differences in susceptibility between IL-17A-deficient (IL-17A-/-) and wild-type (WT) C57BL/6 mice, it was critical for mediating antiviral responses after challenge/reexposure. Compared to WT mice, IL-17A-/- mice (i) infected intravaginally and reexposed or (ii) vaccinated intranasally and challenged intravaginally demonstrated poor outcomes. Following intravaginal HSV-2 reexposure or challenge, vaccinated IL-17A-/- mice had significantly higher mortality, greater disease severity, higher viral shedding, and higher levels of proinflammatory cytokines and chemokines in vaginal secretions. Furthermore, IL-17A-/- mice had impaired Th1 cell responses after challenge/reexposure, with significantly lower proportions of vaginal IFN-γ+ CD4+ T cells. The impaired Th1 cell responses in IL-17A-/- mice coincided with smaller populations of IFN-γ+ CD4+ tissue resident memory T (TRM) cells in the genital tract postimmunization. Taken together, these findings describe a novel role for IL-17A in regulating antiviral IFN-γ+ Th1 cell immunity in the vaginal tract. This strategy could be exploited to enhance antiviral immunity following HSV-2 vaccination.IMPORTANCE T helper type 1 (Th1) immunity, specifically interferon gamma (IFN-γ) production by CD4+ T cells, is critical for protection against genital herpesvirus (HSV-2) infection, and enhancing this response can potentially help improve disease outcomes. Our study demonstrated that interleukin-17A (IL-17A) plays an essential role in enhancing antiviral Th1 responses in the female genital tract (FGT). We found that in the absence of IL-17A, preexposed and vaccinated mice showed poor disease outcomes and were unable to overcome HSV-2 reexposure/challenge. IL-17A-deficient mice (IL-17A-/-) had smaller populations of IFN-γ+ CD4+ tissue resident memory T (TRM) cells in the genital tract postimmunization than did wild-type (WT) mice, which coincided with attenuated Th1 responses postchallenge. This has important implications for developing effective vaccines against HSV-2, as we propose that strategies inducing IL-17A in the genital tract may promote more effective Th1 cell immunity and better overall protection.

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-1ß, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1ß KO, but not IL-6 KO vaginal DCs, showing that IL-1ß is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1ß in vaginal DCs, and addition of IL-1ß restored Th17 induction by IL-1ß 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.].

Medroxyprogesterone Acetate Regulates HIV-1 Uptake and Transcytosis but Not Replication in Primary Genital Epithelial Cells, Resulting in Enhanced T-Cell Infection.

Although clinical and experimental evidence indicates that female sex hormones and hormonal contraceptives regulate susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, the underlying mechanism remains unknown. Genital epithelial cells (GECs) are the first cells to encounter HIV during sexual transmission and their interaction with HIV may determine the outcome of exposure. This is the first report that HIV uptake by GECs increased significantly in the presence of the hormonal contraceptive medroxyprogesterone acetate (MPA) and progesterone and that uptake occurred primarily via endocytosis. No productive infection was detected, but endocytosed virus was released into apical and basolateral compartments. Significantly higher viral transcytosis was observed in the presence of MPA. In GEC and T-cell cocultures, maximum viral replication in T cells was observed in the presence of MPA, which also broadly upregulated chemokine production by GECs. These results suggest that MPA may play a significant role in regulating susceptibility to HIV.

Depot medroxyprogesterone acetate (DMPA) enhances susceptibility and increases the window of vulnerability to HIV-1 in humanized mice.

The progestin-based hormonal contraceptive Depot Medroxyprogesterone Acetate (DMPA) is widely used in sub-Saharan Africa, where HIV-1 is endemic. Meta-analyses have shown that women using DMPA are 40% more likely than women not using hormonal contraceptives to acquire Human Immunodeficiency Virus (HIV-1). Therefore understanding how DMPA increases susceptibility to HIV-1 is an important public health issue. Using C57BL/6 mice and our previously optimized humanized mouse model (NOD-Rag1tm1Mom Il2rgtm1Wjl transplanted with hCD34-enriched hematopoietic stem cells; Hu-mice) where peripheral blood and tissues are reconstituted by human immune cells, we assessed how DMPA affected mucosal barrier function, HIV-1 susceptibility, viral titres, and target cells compared to mice in the diestrus phase of the estrous cycle, when endogenous progesterone is highest. We found that DMPA enhanced FITC-dextran dye leakage from the vaginal tract into the systemic circulation, enhanced target cells (hCD68+ macrophages, hCD4+ T cells) in the vaginal tract and peripheral blood (hCD45+hCD3+hCD4+hCCR5+ T cells), increased the rate of intravaginal HIV-1 infection, extended the window of vulnerability, and lowered vaginal viral titres following infection. These findings suggest DMPA may enhance susceptibility to HIV-1 in Hu-mice by impairing the vaginal epithelial barrier, increasing vaginal target cells (including macrophages), and extending the period of time during which Hu-mice are susceptible to infection; mechanisms that might also affect HIV-1 susceptibility in women.

Medroxyprogesterone acetate alters the vaginal microbiota and microenvironment in women and increases susceptibility to HIV-1 in humanized mice.

The hormonal contraceptive medroxyprogesterone acetate (MPA) is associated with increased risk of human immunodeficiency virus (HIV), via incompletely understood mechanisms. Increased diversity in the vaginal microbiota modulates genital inflammation and is associated with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually transmitted infections (STIs), with Nugent scores <7 (N=58 of 370 screened), MPA correlated with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene sequencing. MPA was also significantly associated with decreased levels of estrogen in the plasma, and low vaginal glycogen and α-amylase, factors implicated in vaginal colonization by lactobacilli, bacteria that are believed to protect against STIs. In a humanized mouse model, MPA treatment was associated with low serum estrogen, low glycogen and enhanced HIV-1 susceptibility. The mechanism by which the MPA-mediated changes in the vaginal microbiota may contribute to HIV-1 susceptibility in humans appears to be independent of inflammatory cytokines and/or activated T cells. Altogether, these results suggest MPA-induced hypo-estrogenism may alter key metabolic components that are necessary for vaginal colonization by certain bacterial species including lactobacilli, and allow for greater bacterial diversity in the vaginal microbiota.This article has an associated First Person interview with the first author of the paper.

Transcriptional profiling of primary endometrial epithelial cells following acute HIV-1 exposure reveals gene signatures related to innate immunity.

PROBLEM: Genital epithelial cells (GECs) line the mucosal surface of the female genital tract (FGT) and are the first cells that interface with both commensal microbiota and sexually transmitted pathogens. Despite the protective barrier formed by GECs, the FGT is a major site of HIV-1 infection. This highlights the importance of studying the interaction of HIV-1 and GECs. METHOD OF STUDY: Using microarray analysis, we characterized the transcriptional profile of primary endometrial GECs grown in the presence or absence of physiological levels of E2 (10-9  mol/L) or P4 (10-7  mol/L) following acute exposure to HIV-1 for 6 hours. RESULTS: Acute exposure of primary endometrial GECs to HIV-1 resulted in the expression of genes related to inflammation, plasminogen activation, adhesion and diapedesis and interferon response. Interestingly, exposure to HIV-1 in the presence of E2 and P4 resulted in differential transcriptional profiles, suggesting that the response of primary endometrial GECs to HIV-1 exposure is modulated by female sex hormones. CONCLUSION: The gene expression signature of endometrial GECs indicates that the response of these cells may be key to determining host susceptibility to HIV-1 and that sex hormones modulate these interactions. This study allows us to explore possible mechanisms that explain the hormone-mediated fluctuation of HIV-1 susceptibility in women.

Frequency of Human CD45+ Target Cells is a Key Determinant of Intravaginal HIV-1 Infection in Humanized Mice.

Approximately 40% of HIV-1 infections occur in the female genital tract (FGT), primarily through heterosexual transmission. FGT factors determining outcome of HIV-1 exposure are incompletely understood, limiting prevention strategies. Here, humanized NOD-Rag1-/- γc-/- mice differentially reconstituted with human CD34+ -enriched hematopoietic stem cells (Hu-mice), were used to assess target cell frequency and viral inoculation dose as determinants of HIV-1 infection following intravaginal (IVAG) challenge. Results revealed a significant correlation between HIV-1 susceptibility and hCD45+ target cells in the blood, which correlated with presence of target cells in the FGT, in the absence of local inflammation. HIV-1 plasma load was associated with viral dose at inoculation and frequency of target cells. Events following IVAG HIV-1 infection; viral dissemination and CD4 depletion, were not affected by these parameters. Following IVAG inoculation, HIV-1 titres peaked, then declined in vaginal lavage while plasma showed a reciprocal pattern. The greatest frequency of HIV-1-infected (p24+) cells were found one week post-infection in the FGT versus blood and spleen, suggesting local viral amplification. Five weeks post-infection, HIV-1 disseminated into systemic tissues, in a dose-dependent manner, followed by depletion of hCD45+ CD3+ CD4+ cells. Results indicate target cell frequency in the Hu-mouse FGT is a key determinant of HIV-1 infection, which might provide a useful target for prophylaxis in women.

High Physiological Concentrations of Progesterone Reverse Estradiol-Mediated Changes in Differentiation and Functions of Bone Marrow Derived Dendritic Cells.

Female sex steroids, estradiol (E2) and progesterone (P4), play a key role in regulating immune responses in women, including dendritic cell (DC) development, and functions. Although the two hormones co-occur in the body of women throughout the reproductive years, no studies have explored their complex combinatorial effects on DCs, given their ability to regulate each other's actions. We examined murine bone marrow derived dendritic cells (BMDC) differentiation and functions, in the presence of a wide range of physiological concentrations of each hormone, as well as the combination of the two hormones. E2 (10(-12) to 10(-8)M) enhanced the differentiation of CD11b+CD11c+ DCs from BM precursor cells, and promoted the expression of CD40 and MHC Class-II, in a dose-dependent manner. In contrast, P4 (10(-9) to 10(-5)M) inhibited DC differentiation, but only at the highest concentrations. These effects on BMDCs were observed both in the presence or absence of LPS. When both hormones were combined, higher concentrations of P4, at levels seen in pregnancy (10(-6)M) reversed the E2 effects, regardless of the concentration of E2, especially in the absence of LPS. Functionally, antigen uptake was decreased and pro-inflammatory cytokines, IL-12, IL-1 and IL-6 production by CD11b+CD11c+ DCs, was increased in the presence of E2 and these effects were reversed by high concentrations of P4. Our results demonstrate the distinct effects of E2 and P4 on differentiation and functions of bone marrow myeloid DCs. The dominating effect of higher physiological concentrations of P4 provides insight into how DC functions could be modulated during pregnancy.

Innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following HIV infection.

The male and female reproductive tracts are complex microenvironments that have diverse functional demands. The immune system in the reproductive tract has the demanding task of providing a protective environment for a fetal allograft while simultaneously conferring protection against potential pathogens. As such, it has evolved a unique set of adaptations, primarily under the influence of sex hormones, which make it distinct from other mucosal sites. Here, we discuss the various components of the immune system that are present in both the male and female reproductive tracts, including innate soluble factors and cells and humoral and cell-mediated adaptive immunity under homeostatic conditions. We review the evidence showing unique phenotypic and functional characteristics of immune cells and responses in the male and female reproductive tracts that exhibit compartmentalization from systemic immunity and discuss how these features are influenced by sex hormones. We also examine the interactions among the reproductive tract, sex hormones and immune responses following HIV-1 infection. An improved understanding of the unique characteristics of the male and female reproductive tracts will provide insights into improving clinical treatments of the immunological causes of infertility and the design of prophylactic interventions for the prevention of sexually transmitted infections.