PROBLEM:The female reproductive tract (FRT) is susceptible to sexually transmitted infections (STIs), including human immunodeficiency virus (HIV), which negatively impact and threaten the lives of women worldwide. While the hormonal regulation of innate and adaptive immunity in the FRT during the menstrual cycle is well-studied, changes in mucosal immune protection throughout the FRT that occur with aging after menopause remain largely unknown. METHODS:This review summarizes hormonal and age-related immune changes throughout the FRT, focusing on their effects on the function of epithelial cells and immune cells in the uterus, cervix and vagina, highlighting implications for HIV infection. RESULTS:After menopause, as reproductive function ceases, two interconnected processes, menopause and immunosenescence, drive alterations in immune protection. As women age following menopause, significant aspects of innate and adaptive immunity in the FRT are compromised in a site-specific manner. At some FRT sites, as cell numbers decline, immunological compensation characterized by increased immunological activity is observed. CONCLUSIONS:Overall, these changes in mucosal immune protection contribute to a heightened risk of STIs and HIV acquisition. Further research is essential to establish a basis for developing new therapeutic interventions to restore immune protection and mitigate conditions that endanger the health and lives of aging women.

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. Unexpectedly, the CD4+ T cell expression of two known correlates of HIV infection susceptibly, CCR5 and integrin-α4β7, increased following menopause across all three tissues despite only being associated with increased infection frequency in EM derived CD4+ T cells. After controlling for changes in the expression of either receptor, both CCR5 and α4β7 expressing CD4+ T cells isolated from the EM of post-menopausal women remained more susceptible to HIV-1 infection than those isolated from pre-menopausal women. Shifts in T helper subset composition, including increases in Th1 frequency and decreases in Th17 and Treg frequency were also observed in the EM only following menopause, but did not correlate with increased infection frequency. Treatment of EM derived CD4+ T cells with 17β-estradiol (E2) prior to viral infection, reduced infection frequency independent of changes in either CCR5 or α4β7 expression frequency. Our results demonstrate that the susceptibility of EM derived CD4+ T cells to HIV-1 infection increases post menopause but is unlikely to be driven by increased expression frequency of either CCR5 or integrin-α4β7. These findings contribute to our understanding of how advanced age alters HIV infection risk which will become increasingly important as the human population continues to age.

ABSTRACTProblemAging alters immune function in women and can lead increased risk of infections, particularly in the female reproductive tract (FRT).Method of StudyTo 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.ResultsConstitutive expression of most PRRs did not vary with age or menopausal status in either cell type. However, the expression of TLR7, MDA5, and NOD2 by ECX stromal fibroblasts significantly increased in post‐menopausal women, while the expression of NOD1 by CX stromal fibroblast also significantly increased in post‐menopausal women. When stratified by age, the expression of TLR6 by CX stromal fibroblasts, and MDA5 and NOD2 by ECX stromal fibroblasts increased significantly with increasing age. Stimulation with the dsRNA viral mimic HMW poly (I:C), a ligand for MDA5, resulted in significantly increased expression of the Type I interferons (IFN) IFNβ and IFNε, the Type III interferon IFNλ1, and interferon‐stimulated genes (ISGs) MxA, OAS2, and ISG15 in both cell populations. However, upregulation of IFNβ, IFNλ1, MxA, OAS2, and ISG15 in response to poly (I:C) significantly declined with increasing post‐menopausal age in ECX stromal fibroblasts. There was no effect of age or menopause on either IFN or ISG expression in CX stromal fibroblasts.ConclusionOverall, these studies demonstrate that ECX and CX fibroblasts are phenotypically distinct populations and that increasing post‐menopausal age reduces IFN and ISG upregulation in ECX stromal fibroblasts in response to viral stimulation, potentially leading to decreased protection against incoming viral pathogens in older post‐menopausal women.

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. We observed a significant decrease in both the total number and percentage of CD4+ T cells in the EM with increasing age, particularly in the years following menopause. CD4+ T cells within the FRT predominantly expressed CD69. The proportion of CD69+CD4+ T cells increased significantly with increasing age in the EM, CX and ECX. The composition of T helper cell subsets within the EM CD4+ T cell population also showed age-related changes. Specifically, there was a significant increase in the proportion of Th1 cells and a significant decrease in Th17 and Treg cells with increasing age. Furthermore, the production of IFNγ by CD4+ T cells in the EM, CX, and ECX significantly decreased with increasing age upon activation. Our findings highlight the complex changes occurring in CD4+ T cell frequency, phenotype, and function within the FRT as women age. Understanding these age-related immune changes in the FRT is crucial for enhancing our knowledge of reproductive health and immune responses in women.

Background Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence. Methods We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples. Results We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1β, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here. Conclusions Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.

had increased capacity to degranulate and secret pro-inflammatory cytokines including IFN ɣ , TNF α , IL-13, IL17 and IL4. Surprisingly, the expression of PD1 was associated with an increased CD8 + Tem functionality. Conclusions : High-dimensional flow cytometric and functional anal-ysis of decidual CD8 + T cells demonstrates that CD8 + T cells have high phenotypic and functional diversity. Here we identi-fied unique dysfunctional and activated cytolytic decidual CD8 + Tem cell types. Understanding how the decidual CD8 + Tem functional heterogeneity relates to their antigen specificity is crucial in advancing our understanding of their contribution to placen-tal inflammation, pregnancy complications and control of congenital infections.

47 its stimulation. It is possible, although not known, that ovarian tumor cells may avoid NK cells either by cleaving MICA/B from its surface or exhausting NK cells through excessive stimulation by tumor-induced IL-15 and induction of CISH expression. It is hypothesized that cleaving of NKG2D ligands and induction of CISH may be associated with prevalence of chronic stress and enhanced GRP78 (glucose regulatory protein 78, a marker of cellular stress) expression in tumor cells. The goal of this studywas to understandmolecularmechanisms induced by ovarian malignant cells to escape NK recognition and/or induction of NK exhaustion by CISH expression. It also examined effects of dietary supplementation with Ashwagandha root powder, an herb, in preventing ovarian tumor-induced suppression of NK cells. Materials and methods: Two experiments were conducted in this study. In the exploratory one (with clinical specimens), normal ovaries from postmenopausal women (55-75years old, n = 10), ovarian malignant tumors at early and late stages (n = 16 from each stage, 4 from each histological subtypes) were used. Localization of MICA, CISH-expressing NK cells, ADAM10 (a protease expressed on tumor cell surface), expression of GRP78 and its regulator miRNA-181a were examined by immunohistochemistry (IHC), immunoblotting (WB) or gene expression studies. In prospective (pre-clinical) one, laying hens (4-year-old) were dietary supplemented with 2% ASH root powder for 120 days. Ovarian tissues from normal (n = 10) or tumor (n = 10 each, supplemented with or without ASH) hens were processed at the end of the study and expression of markers mentioned above were examined. Data from all groups were analyzed using ANOVA and paired or unpaired t-tests and significance were taken when P<0.05. Results: Compared with normal, expression for CISH and MICA was significantly higher (P<0.01) in ovarian tumors. Similarly, ADAM10 and GRP78 expression was also significantly (P<0.001) increased in tumors. Increase in GRP78 expression was associated with the decrease in its regulator miR-181a. Similar to OVCA patients, tumors in hens also showed significant increase in expression of CISH, GRP78, MICA, ADAM10 and decrease in miRNA-181a. Dietary supplementation with ASH showed significant decrease in CISH, GRP78, ADAM10 andMICA (P<0.001) and increase inmiRNA-181a expression. Conclusions: CISH and MICA, two factors related with NK suppression, increased during OVCA development and progression. Increase in CISH and MICA expression was associated with GRP78, a stress marker. ASH, an anti-stress herb, enhanced NK cell function by reducing GRP78 expression. These results suggest that reducing cellular stress may prevent NK cell exhaustion. Support: Swim Across America (AB) S07.4 (Oral Abstract Presentation) SQSTM1/p62 might be involved in radio sensitivity of HPV-infected cervical cancer cells Mihoko Kawaguchi, Atsushi Furuta, Akemi Yamaki, Ippei Yasuda, Kyoko Takemura, Tomoko Shima, Akitoshi Nakashima Toyama University, Toyama, Japan Problem: Approximately 570,000 women are diagnosed with cervical cancer each year all over the world, and about 311,000 women died from it. Most cervical cancers are caused by human papillomavirus (HPV) infection. It has been reported that HPV infection suppresses autophagy in cervical epithelial cells, resulting in the accumulation of SQSTM1/p62 (p62), which might be favorable for the viral growth and cervical cancer progression. Radiotherapy is generally considered as a central treatment of cervical cancer. To date, however, adjuvant therapy targeting p62 has not been established. In this study, we conducted an experiment to investigate whether the expression level of p62 affects the sensitivity of radiotherapy in the cervical cancer cell lines. Methods of Study: We evaluated p62 expression in HeLa cells (infected with HPV type 18), ME180 (HPV type 68) and C33A (HPV negative) by western blot. We also compared p62 expression in cervical cancer and dysplasia tissues by immunohistochemistry. Radiation was applied at an absorbed dose of 6 Gy or 12 Gy, and the survival rate after irradiation was evaluated at 48 hours post-irradiation by WST1 assay. In addition, knock down of p62 was conducted by transducing p62-siRNA. The effect of siRNA was estimated after 48 hours of the transduction. Results: The expression of p62 was higher in HeLa and ME180, HPVinfected cells, compared with C33A, non-infected cells. Immunohistochemical analysis showed that p62 staining was stronger in cervical cancer tissues than in CIN2 and CIN3 tissues. When HeLa andME180 cells were irradiated, the cell viability was decreased dependently of radiation dose. The viability of cells irradiated with 12 Gy was 20% lower in HeLa cells and 25% lower in ME180 cells than that of nonirradiated cells. On the other hand, there was stable in the viable cell rate in C33A cells even after 12 Gy irradiation. We confirmed that siRNA transfection knocked down p62 expression by 76%, 84%, and 91% in HeLa, ME180, and C33A, respectively. In the p62-knockdown ME180, HPV-infected cells, irradiation-induced cellular growth inhibition was attenuated by p62 downregulation after 12 Gy irradiation. In addition, there was no decrease in the viable cell rate of HPVuninfected cells, whose p62 was downregulated by siRNA, after irradiation with or without knockdown. Conclusions: HPV-infected cervical cancer cells were more radiosensitive than uninfected cells. The radiotherapeutic sensitivity might be related with the p62 expression level in the HPV-positive cervical cancer. Increasing p62 expression would be a target to improve radiosensitivity of cervical cancer. S07.5 (Oral Abstract Presentation) Persistent exposure to ovarian tumor-induced IL-15 leads to exhaustion of NK cells through induction of cytokine-induced SH2 (CISH) containing protein Jasmin C Acosta, Pincas Bitterman, Animesh Barua Rush University Medical Center, Chicago, IL, USA Problem: Due to heterogeneity in origin and the lack of an effective early detection test, ovarian cancer (OVCA) in most cases is detected

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. Using hysterectomy EM tissues, we found that EM CD8+ T cell numbers declined significantly in the years following menopause. Despite an overall decline in CD8+ T cells, cytotoxic activity per cell for both CD103- and CD103 + CD8+ T cells increased with age. Investigation of the underlying mechanisms responsible for cytotoxicity indicated that the percentage of total granzyme A and granzyme B positive CD8+ T cells, but not perforin, increased significantly after menopause and remained high and constant as women aged. Additionally, baseline TNFα production by EM CD8+ T cells increased significantly in the years following menopause, and estradiol suppressed TNFα secretion. Moreover, in response to PMA activation, TNFα and IFNγ were significantly up-regulated, and CD103-CD8+ T cells up-regulation of TNFα, IFNγ and IL-6 increased as women aged. Understanding the underlying factors involved in regulating cell-mediated protection of the EM by CD8+ T cells will contribute to the foundation of information essential for developing therapeutic tools to protect women against gynecological cancers and infections as they age.

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. The endometrial immune system is essential for generating a receptive endometrium, but the link between the immune and reproductive systems in the endometrium in the years prior to menopause has not been well-defined. Since the endometrial immune system is tightly regulated to maximize reproductive success and pathogen protection, changes in immune function with increasing premenopausal age have the potential to impact reproduction.

mechanisms associated with failure of IDO1 blockade, we conducted a pilot, window-of-opportunity clinical study testing the immunological and metabolic effects of an IDO1 inhibitor in patients with newly diagnosed advanced high grade serous ovarian cancer prior to their standard tumor debulking surgery. We showed efficient blockade of the kynurenine pathway of tryptophan degradation with the ovarian tumormicroenvironment.However, this blockadewas accompanied by a metabolic adaptation that shunted tryptophan catabolism towards the serotoninpathwayandelevatednicotinamideadeninedinucleotide (NAD)+ biosynthetic pathways, whichwas detrimental for T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function. We demonstrated that A2a and A2b, or the combination ofA2a andA2bpurinergic receptor antagonists rescuedNAD+mediated suppression of T cell proliferation and function, and the combination of IDO1 inhibition and A2a/A2b receptor blockade improved tumor immune signature and survival in an IDO1 over-expressing preclinical mouse model of ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade that may undermine efforts to induce tumor-specific T cell responses.

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. MPA significantly impaired wound closure in both cell types and delayed the reestablishment of transepithelial resistance by epithelial cells. In contrast to MPA, NET selectively decreased wound closure by stromal fibroblasts but not epithelial cells. Following epithelial injury, MPA but not LNG or NET, blocked the injury-induced upregulation of HBD2, a broad-spectrum antimicrobial implicated in wound healing, but had no effect on the secretion of RANTES, CCL20 and SDF-1α. This study demonstrates that, unlike progesterone and LNG, MPA and NET may interfere with wound closure following injury in the endometrium, potentially conferring a higher risk of pathogen transmission. Our findings highlight the importance of evaluating progestins for their impact on wound repair at mucosal surfaces.

Estradiol (E2) 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 E2 and P levels. However, the effect of E2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E2 (5x10-8M) or P (1x10-7M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E2, P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E2 and P on IFNλ1-induced ISGs are cell-type specific. E2-mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.

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. After menopause, natural reproductive function ends, and therefore, two overlapping processes contribute to alterations in immune protection in aging women: menopause and immunosenescence. The goal of this review is to summarize the multiple immune changes that occur in the FRT with aging, including the impact on the function of epithelial cells, immune cells, and stromal fibroblasts. These studies indicate that major aspects of innate and adaptive immunity in the FRT are compromised in a site-specific manner in the FRT as women age. Further, at some FRT sites, immunological compensation occurs. Overall, alterations in mucosal immune protection contribute to the increased risk of sexually transmitted infections (STI), urogenital infections, and gynecological cancers. Further studies are essential to provide a foundation for the development of novel therapeutic interventions to restore immune protection and reverse conditions that threaten women's lives as they age.

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. Tumor CD8+ T cells expressed significantly less granzyme A (GZA), B (GZB), and PD-1 than those in adjacent non-cancerous tissues and also had significantly lower cytotoxic killing of allogeneic target cells. CD103-CD8+ T cells, but not CD103+CD8+ T cells, from both adjacent and tumor tissue were primarily responsible for killing of allogeneic target cells. Secretions recovered from endometrial carcinoma tissues suppressed CD8+ cytotoxic killing and lowered perforin, GZB and PD-1 expression relative to non-tumor CD8+ T cells. Furthermore, tumor secretions contained significantly higher levels of immunosuppressive cytokines including TGFβ than non-tumor tissues. Thus, the tumor microenvironment suppresses cytotoxic killing by CD8+ T cells via the secretion of immunosuppressive cytokines leading to decreased expression of intracellular cytolytic molecules. These studies demonstrate the complexity of CD8+ T cell regulation within the endometrial tumor microenvironment and provide a foundation of information essential for the development of therapeutic strategies for gynecological cancers.

The human female reproductive tract (FRT) is a unique mucosal surface with two competing functions: creating an immunological environment conducive to successful reproduction while maintaining protection against pathogens from the external environment that lead to increased reproductive dysfunction, morbidity, and mortality in women. Key to this unique balancing act are the sex hormones estradiol and progesterone whose concentrations undergo cyclical changes in premenopausal women and regulate multiple aspects of innate and adaptive immunity thus leading to changes in immune protection across the menstrual cycle. Defining the composition and regulation of the FRT immune system are essential in understanding how this unique mucosal surface accommodates both reproduction and immune protection.

Immune protection in the female reproductive tract (FRT) has evolved to meet the challenges of sexually transmitted bacterial and viral pathogens, allogeneic spermatozoa, and an immunologically distinct semi-allogeneic fetus. Throughout the FRT, the innate immune system is essential for the recognition and initial response to incoming pathogens. Key mediators of innate immune protection examined in this review include epithelial cells, stromal fibroblasts, macrophages, DC, and neutrophils from the Fallopian tubes, uterus, cervix and vagina. These innate immune cells respond to pathogens resulting in the secretion of cytokines, chemokines, antimicrobials, and production of intracellular proteins that protect, activate and recruit both innate and adaptive immune cells. Human immunodeficiency virus (HIV) infection can occur throughout the FRT, including the ovary, and is modulated by multiple factors including age of the individual, epithelial barrier integrity, composition of the vaginal microbiome, and hormonal status. Alterations in immune function due to hormonal changes that optimize conditions for successful fertilization create a hypothesized “window of vulnerability” that lasts from ovulation into the secretory stage of the menstrual cycle. The goal of this review is to summarize the multiple levels of protection against HIV infection in the FRT and thereby providing a foundation for the design of vaccines for protection against sexually-transmitted infections (STI) including HIV.

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. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

ProblemThe contribution of fibroblasts to innate immune protection of the human female reproductive tract (FRT) against viral pathogens is relatively unknown.Method of StudyEndometrial (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 (E2), with gene expression measured by real‐time RT‐PCR and protein secretion by ELISA.ResultsPoly (I:C) induced the expression of the interferon‐stimulated genes (ISG) MxA, OAS2 and APOBEC3G, and the cytokines MCP‐1, IL‐8, IL‐6, CCL20, IFNβ and RANTES by fibroblasts from all three sites. ISG upregulation was dependent upon Type I IFN signaling. E2 inhibited the poly (I:C)‐induced upregulation of MxA and OAS2 in EM fibroblasts, but not Cx or ECx fibroblasts. E2 upregulated SDF‐1α by EM fibroblasts but had no effect on secretion of other cytokines either alone or in the presence of poly (I:C). Conditioned media (CM) from poly (I:C)‐treated or E2‐treated fibroblasts significantly reduced HIV infection of CD4+ T cells.ConclusionStromal fibroblasts represent a level of innate immune protection against viral pathogens in the FRT beyond that seen with epithelial cells and immune cells. Our findings indicate that fibroblasts FRT are selectively responsive to E2, capable of initiating an antiviral response against viral pathogens and may play a role in preventing HIV infection of CD4+ T cells.

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. Poly (I:C) and LPS, respective mimics of viral and bacterial infections, selectively upregulated antiviral gene expression and secretion of chemokines and antimicrobials. Poly (I:C) exclusively stimulated the expression of interferon (IFN) beta, IFN lambda 1, and the IFN-stimulated gene OAS2. Poly (I:C) also significantly increased secretion of elafin, CCL20, and RANTES, but had no effect on SDF-1 alpha. In contrast, LPS had no effect on IFN or ISG expression but significantly increased secretion of RANTES and SDF-1 alpha. Secretions from poly (I:C)-treated fibroblasts had both greater anti-HIV activity and induced higher levels of CD4 + T cell chemotaxis than those from LPS-treated cells. Our studies demonstrate a potential key role for ovarian fibroblasts in innate immune protection against incoming pathogens in the normal ovary.