Increases in the susceptibility of human endometrial CD4 T cells to HIV-1 infection post-menopause are not dependent on greater viral receptor expression frequency.

Epidemiological evidence suggests that post-menopausal women are more susceptible to HIV infection following sexual intercourse than are younger cohorts for reasons that remain unclear. Here, we evaluated how menopause-associated changes in CD4 T cell numbers and subsets as well as HIV coreceptor expression, particularly CCR5, in the endometrium (EM), endocervix (CX), and ectocervix (ECX) may alter HIV infection susceptibility. Using a tissue-specific mixed cell infection model, we demonstrate that while no changes in CD14 macrophage infection susceptibility were observed, CD4 T cell HIV-1 infection frequency increases following menopause in the EM, but not CX nor ECX.

Aging Selectively Alters PRR and ISG Expression in Endo- and Ecto-Cervical Stromal Fibroblasts From the Human Female Reproductive Tract.

Problem: Aging alters immune function in women and can lead increased risk of infections, particularly in the female reproductive tract (FRT).

Method Of Study: To determine how aging affects innate immune responses in the cervical stroma of the FRT, we isolated endocervical (CX) and ectocervical (ECX) stromal fibroblasts and determine if their expression of multiple pattern recognition receptors (PRRs) and responses to viral stimulation varied with menopause and age.

Results: Constitutive expression of most PRRs did not vary with age or menopausal status in either cell type.

Impact of aging on the frequency, phenotype, and function of CD4+ T cells in the human female reproductive tract.

Since CD4+ T cells are essential for regulating adaptive immune responses and for long lasting mucosal protection, changes in CD4+ T cell numbers and function are likely to affect protective immunity. What remains unclear is whether CD4+ T cell composition and function in the female reproductive tract (FRT) changes as women age. Here we investigated the changes in the composition and function of CD4+ T cells in the endometrium (EM), endocervix (CX), and ectocervix (ECX) with aging.

Aging beyond menopause selectively decreases CD8+ T cell numbers but enhances cytotoxic activity in the human endometrium.

Background: Regulation of endometrial (EM) CD8+ T cells, which provide protection through cell-mediated cytotoxicity, is essential for successful reproduction, and protection against sexually transmitted infections and potential tumors. We have previously demonstrated that EM CD8+ T cell cytotoxicity is suppressed directly and indirectly by sex hormones and enhanced after menopause. What remains unclear is whether CD8+ T cell protection and the contribution of tissue-resident (CD103+) and non-resident (CD103-) T cell populations in the EM change as women age following menopause.

Do endometrial immune changes with age prior to menopause compromise fertility in women?

Menopause signals the end of the reproductive period in women. However, fertility and fecundity decrease with increasing age prior to menopause demonstrating that changes in the premenopausal female reproductive tract (FRT) are already occurring that negatively impact reproductive success. The effects of age on the endometrium are poorly understood, in contrast to the ovary where changes occur with increasing age that negatively affect successful reproduction.

Medroxyprogesterone acetate inhibits wound closure of human endometrial epithelial cells and stromal fibroblasts in vitro.

Mucosal integrity in the endometrium is essential for immune protection. Since breaches or injury to the epithelial barrier exposes underlying tissue and is hypothesized to increase infection risk, we determined whether endogenous progesterone or three exogenous progestins (medroxyprogesterone acetate (MPA), norethindrone (NET), and levonorgestrel (LNG)) used by women as contraceptives interfere with wound closure of endometrial epithelial cells and fibroblasts in vitro. Progesterone and LNG had no inhibitory effect on wound closure by either epithelial cells or fibroblasts.

Sex Hormones and Aging Modulate Interferon Lambda 1 Production and Signaling by Human Uterine Epithelial Cells and Fibroblasts.

Estradiol (E) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E and P levels. However, the effect of E and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown.

Endometrial Cancer Suppresses CD8+ T Cell-Mediated Cytotoxicity in Postmenopausal Women.

Endometrial cancer is the most common gynecological cancer. To investigate how it suppresses host immune function, we isolated CD8+ T cells from endometrial endometroid carcinomas and adjacent non-cancerous endometrium and determined if the tumor environment regulates cytotoxic capacity. Endometrial carcinomas had increased numbers of CD8+ T cells compared to adjacent non-cancerous endometrium.

The impact of aging on innate and adaptive immunity in the human female genital tract.

Mucosal tissues in the human female reproductive tract (FRT) are primary sites for both gynecological cancers and infections by a spectrum of sexually transmitted pathogens, including human immunodeficiency virus (HIV), that compromise women's health. While the regulation of innate and adaptive immune protection in the FRT by hormonal cyclic changes across the menstrual cycle and pregnancy are being intensely studied, little to nothing is known about the alterations in mucosal immune protection that occur throughout the FRT as women age following menopause. The immune system in the FRT has two key functions: defense against pathogens and reproduction.

Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells.

Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells.

Estradiol-regulated innate antiviral responses of human endometrial stromal fibroblasts.

Problem: The contribution of fibroblasts to innate immune protection of the human female reproductive tract (FRT) against viral pathogens is relatively unknown.

Method Of Study: Endometrial (EM), endocervical (Cx) and ectocervical (ECx) fibroblasts were isolated from hysterectomy patients and grown in vitro. Fibroblasts were treated with the viral mimic poly (I:C) in the presence or absence of the sex hormone estradiol (E ), with gene expression measured by real-time RT-PCR and protein secretion by ELISA.

Poly (I:C) and LPS induce distinct immune responses by ovarian stromal fibroblasts.

Despite its anatomical location, the ovary is a site of pathogen exposure in the human female reproductive tract (FRT). However, the role of ovarian stromal fibroblasts in immune protection is unclear. We generated a population of ovarian stromal fibroblasts derived from normal human ovaries that expressed the pattern recognition receptors TLR3, TLR4, RIG-I, & MDA5.

IL-27 Expression and Responsiveness in Human Uterine Epithelial Cells and Fibroblasts In Vitro and the Role of Estradiol.

Interleukin (IL)-27 is a pleiotropic cytokine that regulates multiple aspects of innate and adaptive immunity, but whose role in immune protection of the female reproductive tract is unknown. Although not constitutively expressed by human uterine epithelial cells and fibroblasts in culture, IL-27 secretion was upregulated after treatment with the viral ligand poly (I:C) in a type I interferon (IFN)-dependent manner, with higher levels measured in fibroblasts than epithelial cells. Estradiol increased poly (I:C)-induced IL-27 production by fibroblasts, but not epithelial cells.

Hormonal Contraceptives Differentially Suppress TFV and TAF Inhibition of HIV Infection and TFV-DP in Blood and Genital Tract CD4+ T cells.

HIV prevention research is focused on combining antiretrovirals (ARV) and progestin contraceptives to prevent HIV infection and pregnancy. The possibility that progestins compromise ARV anti-HIV activity prompted us to evaluate the effects of progestins on tenofovir (TFV) and TFV-alafenamide (TAF) on HIV infection and intracellular TFV-diphosphate (TFV-DP) concentrations in blood and genital CD4+ T cells. Following incubation of blood CD4+ T cells with TFV or TAF, Medroxyprogesterone acetate (MPA), but not Levonorgestrel, Norethisterone or progesterone, suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP.

Tenofovir Inhibits Wound Healing of Epithelial Cells and Fibroblasts from the Upper and Lower Human Female Reproductive Tract.

Disruption of the epithelium in the female reproductive tract (FRT) is hypothesized to increase HIV infection risk by interfering with barrier protection and facilitating HIV-target cell recruitment. Here we determined whether Tenofovir (TFV), used vaginally in HIV prevention trials, and Tenofovir alafenamide (TAF), an improved prodrug of TFV, interfere with wound healing in the human FRT. TFV treatment of primary epithelial cells and fibroblasts from the endometrium (EM), endocervix (CX) and ectocervix (ECX) significantly delayed wound closure.

Menopausal status influences the expression of programmed death (PD)-1 and its ligand PD-L1 on immune cells from the human female reproductive tract.

Problem: The programmed death 1 (PD-1)/PD-L1 pathway regulates peripheral tolerance, immune responses, and is up-regulated in chronic viral infections, including HIV infection. However, expression of PD-1/PD-L1 on immune cells from the human female reproductive tract (FRT) and possible regulation by menopause and sex hormones are poorly understood.

Method Of Study: PD-1/PD-L1 expression was analyzed on CD4(+) and CD8(+) T cells, CD163(+) macrophages, and CD11c(+) dendritic cells (DC) from endometrium (EM), endocervix (CX) and ectocervix (ECX).

The role of sex hormones in immune protection of the female reproductive tract.

Within the human female reproductive tract (FRT), the challenge of protection against sexually transmitted infections (STIs) is coupled with the need to enable successful reproduction. Oestradiol and progesterone, which are secreted during the menstrual cycle, affect epithelial cells, fibroblasts and immune cells in the FRT to modify their functions and hence the individual's susceptibility to STIs in ways that are unique to specific sites in the FRT. The innate and adaptive immune systems are under hormonal control, and immune protection in the FRT varies with the phase of the menstrual cycle.

Innate immunity in the vagina (Part II): Anti-HIV activity and antiviral content of human vaginal secretions.

Problem: Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions change across the menstrual cycle is unknown.

Method Of Study: Using a menstrual cup, vaginal secretions from pre-menopausal women were recovered at the proliferative (d6-8), mid-cycle (d13-15), and secretory (d21-23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line.

Regulation of mucosal immunity in the female reproductive tract: the role of sex hormones in immune protection against sexually transmitted pathogens.

The immune system in the female reproductive tract (FRT) does not mount an attack against human immunodeficiency virus (HIV) or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the FRT. Working together, these antimicrobials along with mucosal antibodies attack viral, bacterial, and fungal targets.

Estradiol regulation of nucleotidases in female reproductive tract epithelial cells and fibroblasts.

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues.

Estradiol reduces susceptibility of CD4+ T cells and macrophages to HIV-infection.

The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4(+) T-cells and macrophages.

Innate and adaptive anti-HIV immune responses in the female reproductive tract.

The mucosal surface of the female reproductive tract (FRT) is the primary site of transmission for a plethora of sexually transmitted infections, including human immunodeficiency virus (HIV), that represent a significant burden upon womens' health worldwide. However, fundamental aspects of innate and adaptive immune protection against HIV infection in the FRT are poorly understood. The FRT immune system is regulated by the cyclical changes of the sex hormones estradiol and progesterone across the menstrual cycle, which as we have hypothesized, leads to the creation of a window of vulnerability during the secretory stage of the menstrual cycle, when the risk of HIV transmission is increased.

Innate immunity in the vagina (part I): estradiol inhibits HBD2 and elafin secretion by human vaginal epithelial cells.

Problem: Vaginal epithelial cells (VEC) are the first line of defense against incoming pathogens in the female reproductive tract. Their ability to produce the anti-HIV molecules elafin and HBD2 under hormonal stimulation is unknown.

Method Of Study: Vaginal epithelial cells were recovered using a menstrual cup and cultured overnight prior to treatment with estradiol (E₂), progesterone (P₄) or a panel of selective estrogen response modulators (SERMs).

Uterine epithelial cells specifically induce interferon-stimulated genes in response to polyinosinic-polycytidylic acid independently of estradiol.

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR.