Estradiol Enhances Antiviral CD4+ Tissue-Resident Memory T Cell Responses following Mucosal Herpes Simplex Virus 2 Vaccination through an IL-17-Mediated Pathway.

Estradiol (E2) is a sex hormone which has been shown to be protective against sexually transmitted infections such as herpes simplex virus 2 (HSV-2). However, few studies have examined the underlying mechanisms by which this occurs. Here, we investigated the effect of E2 on the establishment of memory T cells post-intranasal immunization with HSV-2. CD4+ T cell responses first appeared in the upper respiratory tract (URT) within 3 days postimmunization before being detected in the female reproductive tract (FRT) at 7 days. E2 treatment resulted in greater and earlier Th17 responses, which preceded augmented Th1 responses at these sites. The CD4+ T cells persisted in the URT for up to 28 days, and E2 treatment resulted in higher frequencies of memory T cells. Intranasal immunization also led to the establishment of CD4+ tissue-resident memory T cells (TRM cells) in the FRT, and E2 treatment resulted in increased Th1 and Th17 TRM cells. When the migration of circulating T cells into the FRT was blocked by FTY720, immunized E2-treated mice remained completely protected against subsequent genital HSV-2 challenge compared to non-E2 controls, confirming that TRM cells alone are adequate for protection in these mice. Finally, the enhanced vaginal Th1 TRM cells present in E2-treated mice were found to be modulated through an interleukin 17 (IL-17)-mediated pathway, as E2-treated IL-17A-deficient mice had impaired establishment of Th1 TRM cells. This study describes a novel role for E2 in enhancing CD4+ memory T cells and provides insight on potential strategies for generating optimal immunity during vaccination.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater Th17 memory cells, which precede enhanced Th1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.

Curcumin Can Decrease Tissue Inflammation and the Severity of HSV-2 Infection in the Female Reproductive Mucosa.

Herpes Simplex Virus Type 2 (HSV-2) is one of the most prevalent sexually transmitted viruses and is a known risk factor for HIV acquisition in the Female Genital Tract (FGT). Previously, we found that curcumin can block HSV-2 infection and abrogate the production of inflammatory cytokines and chemokines by genital epithelial cells in vitro. In this study, we investigated whether curcumin, encapsulated in nanoparticles and delivered by various in vivo routes, could minimize inflammation and prevent or reduce HSV-2 infection in the FGT. Female mice were pre-treated with curcumin nanoparticles through oral, intraperitoneal and intravaginal routes, and then exposed intravaginally to the tissue inflammation stimulant CpG-oligodeoxynucleotide (ODN). Local intravaginal delivery of curcumin nanoparticles, but not intraperitoneal or oral delivery, reduced CpG-mediated inflammatory histopathology and decreased production of pro-inflammatory cytokines Interleukin (IL)-6, Tumor Necrosis Factor Alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP-1) in the FGT. However, curcumin nanoparticles did not demonstrate anti-viral activity nor reduce tissue pathology when administered prior to intravaginal HSV-2 infection. In an alternative approach, intravaginal pre-treatment with crude curcumin or solid dispersion formulations of curcumin demonstrated increased survival and delayed pathology following HSV-2 infection. Our results suggest that curcumin nanoparticle delivery in the vaginal tract could reduce local tissue inflammation. The anti-inflammatory properties of curcumin delivered to the vaginal tract could potentially reduce the severity of HSV-2 infection and decrease the risk of HIV acquisition in the FGT of women.

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.].

The Role of IL-17 During Infections in the Female Reproductive Tract.

Interleukin-17 (IL-17A) is a cytokine involved in a complex array of both protective and detrimental processes. Although early biological studies focused on the pro-inflammatory function of IL-17 in the context of autoimmune and inflammatory disorders, it has become increasingly evident that the roles of IL-17 are far more nuanced. Recent work has demonstrated that the functions of IL-17 are highly context- and tissue-dependent, and there is a fine balance between the pathogenic and protective functions of IL-17. This is especially evident in mucosal tissues such as the female reproductive tract, where IL-17 has been shown to play an important role in the immune response generated during fungal, bacterial and viral infections associated with protection, but also with inflammation. In this review, we discuss the evolving landscape of IL-17 biology within the context of the vaginal mucosa, focusing on key findings that highlight the importance of this cytokine in genital mucosal immunity.

Transcriptional response of vaginal epithelial cells to medroxyprogesterone acetate treatment results in decreased barrier integrity.

Medroxyprogesterone acetate (MPA) is a frequently used hormonal contraceptive that has been shown to significantly increase HIV-1 susceptibility by approximately 40 %. However, the underlying mechanism by which this occurs remains unknown. Here, we examined the biological response to MPA by vaginal epithelial cells, the first cells to encounter HIV-1 during sexual transmission, in order to understand the potential mechanism(s) of MPA-mediated increase of HIV-1 infection. Using microarray analysis and in vitro assays, we characterized the response of vaginal epithelial cells, grown in biologically relevant air-liquid interface (ALI) cultures, to physiological levels of female sex hormones, estradiol (E2), progesterone (P4), or MPA. Transcriptional profiling of E2, P4 or MPA-treated vaginal epithelial cells indicated unique transcriptional profiles associated with each hormone. MPA treatment increased transcripts of genes related to cholesterol/sterol synthesis and decreased transcripts related to cell division and cell-cell adhesion, results not seen with E2 or P4 treatments. MPA treatment also resulted in unique gene expression indicative of decreased barrier integrity. Functional assays confirmed that MPA, but not E2 or P4 treatments, resulted in increased epithelial barrier permeability and inhibited cell cycle progression. The effects of MPA on vaginal epithelial cells seen in this study may help explain the increase of HIV-1 infection in women who use MPA as a hormonal contraceptive.

Cigarette smoke exposure attenuates the induction of antigen-specific IgA in the murine upper respiratory tract.

The upper respiratory tract is highly exposed to airborne pathogens and serves as an important inductive site for protective antibody responses, including mucosal IgA and systemic IgG. However, it is currently unknown to what extent inhaled environmental toxins, such as a cigarette smoke, affect the ability to induce antibody-mediated immunity at this site. Using a murine model of intranasal lipopolysaccharide and ovalbumin (LPS/OVA) immunization, we show that cigarette smoke exposure compromises the induction of antigen-specific IgA in the upper airways and systemic circulation. Deficits in OVA-IgA were observed in conjunction with a reduced accumulation of OVA-specific IgA antibody-secreting cells (ASCs) in the nasal mucosa, inductive tissues (NALT, cervical lymph nodes, spleen) and the blood. Nasal OVA-IgA from smoke-exposed mice also demonstrated reduced avidity during the acute post-immunization period in association with an enhanced mutational burden in the cognate nasal Igha repertoire. Mechanistically, smoke exposure attenuated the ability of the nasal mucosa to upregulate VCAM-1 and pIgR, suggesting that cigarette smoke may inhibit both nasal ASC homing and IgA transepithelial transport. Overall, these findings demonstrate the immunosuppressive nature of tobacco smoke and illustrate the diversity of mechanisms through which this noxious stimulus can interfere with IgA-mediated immunity in the upper airways.

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.

IL-17 Production by γδ+ T Cells Is Critical for Inducing Th17 Responses in the Female Genital Tract and Regulated by Estradiol and Microbiota.

IL-17 can be produced by adaptive immune cells such as Th17 cells and by immune cells that produce IL-17 without prior priming. This latter category, which we will refer to as "innate," includes innate cells such as NK cells and innate lymphoid cells and innate-like T cell populations such as NKT cells and γδ+ T cells. Studies in mucosal tissues have shown that the induction of Th17 immunity is amplified by innate IL-17 produced within those tissues. However, the role of innate IL-17 and its effect on Th17 induction in the female genital tract (FGT) is largely unknown. In this study, we characterize the primary source of IL-17-secreting vaginal cells and show that innate IL-17 plays a critical role in priming adaptive Th17 responses in the FGT. Under homeostatic conditions, γδ+ T cells were the predominant source of innate IL-17 in the murine FGT, and this population was modulated by both the sex hormone estradiol and the presence of commensal microbiota. Compared with wild-type C57BL/6 mice, vaginal APCs isolated from IL-17A-deficient (IL-17A-/- ) mice were severely impaired at priming Th17 responses in APC-T cell cocultures. Furthermore, the defect in Th17 induction in the absence of innate IL-17 was associated with impairment of IL-1ß production by vaginal CD11c+ dendritic cells. Overall, our study describes a novel role for IL-17 in the FGT and further demonstrates the importance of factors in the vaginal microenvironment that can influence adaptive immune responses.