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

The main cell types in the FRT that have immune capabilities are epithelial cells, stromal fibroblasts and leukocytes. Epithelial cells line the surface of the FRT, providing a barrier that separates the lumen from the underlying tissue ( FIG. 1 ). Multi-layered squamous epithelial cells cover the lower FRT (vagina and ectocervix), whereas single-layer columnar epithelial cells cover the upper FRT (endocervix, uterus and Fallopian tubes). Beneath the epithelium is a dense layer of stromal fibroblasts, which provides structural tissue support. Distributed throughout the stroma is a dynamic population of leukocytes. These account for 6–20% of total cells in the human FRT, with more leukocytes being present in the upper tract than in the lower portions of the tract 5 . Most leukocyte subsets have a preferential distribution within the different sites in the FRT; for example, T cells (CD3 + ), which are the most abundant leukocyte subset in the FRT, have higher proportions in the lower than in the upper tract, whereas granulocytes (CD66b + ) and natural killer (NK) cells are more abundant in the upper tract than in the lower tract ( FIG. 3 ). Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), are essential for the initial detection and response to pathogens as they recognize conserved pathogen-associated molecular patterns (PAMPs). For example, TLR7 and RIG-I recognize HIV, whereas TLR2 and TLR4 recognize C. trachomatis and TLR2 recognizes N. gonorrhoeae 6 – 11 . PRR expression varies within the FRT 12 . Expression of the bacterial receptors TLR2, TLR4, nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 is highest in the upper FRT and declines in the lower FRT, which suggests that the lower FRT might minimize responses against commensal bacteria, whereas the upper tract is very sensitive to bacterial pathogens 13 , 14 . A similar trend is seen for the cytoplasmic PRRs RIG-I and melanoma differentiation-associated protein 5 (MDA5; also known as IFIH1) 14 . By contrast, TLR7, TLR8 and TLR9 are evenly expressed throughout the FRT from the Fallopian tubes to the ectocervix, which suggests that immune recognition of viruses is fairly constant between the upper and the lower FRT 15 . Similar to other aspects of immune protection, PRR expression changes across the menstrual cycle and with hormone exposure. TLR2, TLR6, TLR9 and TLR10 expression is lower in human endometrial tissue recovered at the proliferative phase than in that recovered at the secretory phase 16 – 19 . OE 2 decreases TLR4 mRNA expression by uterine fibroblasts and decreases TLR2 and TLR6 expression by the VK2 vaginal epithelial cell line in vitro , but it has no effect on the expression of other PRRs 20 . P4 increases TLR4 expression by fibroblasts, which suggests that these cells are more sensitive to bacterial pathogens in the secretory phase 17 . However, it has not been directly shown that the levels of PRR expression correlate with protection against pathogens in the FRT. OE 2 also modulates the signalling pathways downstream of PRRs and pro-inflammatory receptors. It inhibits the lipopolysaccharide (LPS)- and polyinosinic–polycytidylic acid (poly(I:C))-induced secretion of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6) and IL-8 by uterine epithelial cells and reverses the stimulatory effects of IL-1β on mRNA and protein expression of tumour necrosis factor (TNF), human β-defensin 2 (HBD2), IL-8 and nuclear factor-κB (NF-κB). This suggests that inflammatory responses to pathogens are decreased during periods of high OE 2 levels in the menstrual cycle 21 , 22 . OE 2 regulates the function of NF-κB, which is a key transcription factor involved in inflammatory gene expression, by restricting its cytoplasmic-to-nuclear translocation or by preventing the degradation of NF-κB inhibitors 23 , 24 . Furthermore, as secretory leukocyte protease inhibitor (SLPI; also known as antileukoproteinase) inhibits NF-κB expression, OE 2 -mediated inhibition of pro-inflammatory cytokine expression may be mediated through the regulation of NF-κB by OE 2 -induced SLPI 25 . Thus, OE 2 may reduce susceptibility to HIV infection in the FRT by creating an anti-inflammatory environment that is characterized by reduced target cell migration as a result of the decreased secretion of inflammatory cytokines, as well as by eliminating the immune-activated environment that is often associated with infections. The lumen of the entire FRT is bathed in fluid, the composition of which differs both between the upper and the lower tract and across the menstrual cycle, and which represents the combined secretions of the different cell types in the FRT. Contained within the fluid are various immunomodulatory molecules including cytokines, chemokines, antimicrobial proteins, enzymes and growth factors. In cervico-vaginal lavage fluid (CVL fluid), the concentrations of antimicrobial proteins such as SLPI, HBD2, human neutrophil peptide 1 (HNP1; also known as neutrophil defensin 1), HNP2, HNP3, lysozyme, lactoferrin and surfactant A markedly decrease by mid-cycle (day 13) and remain low for 7–10 days during the secretory phase before returning to the higher levels found during the proliferative phase following menstruation 26 , 27 . These findings suggest that the antimicrobial contribution of the luminal fluid to overall immune protection in the FRT decreases during the secretory phase. Interestingly, total protein and transforming growth factor-β (TGFβ) levels remain unchanged throughout the cycle, which shows the selectivity of hormone effects. However, other studies have found no changes in secreted levels of various proteins at mid-cycle, possibly as a result of cycle length variation ( BOX 1 ) and differences in sampling technique 28 – 32 . By contrast, IL-6 and IL-1β levels increase during the proliferative phase of the cycle, which shows that concentration changes are specific to certain molecules at specific phases of the cycle 29 . Secretions from the upper FRT have a distinct proteomic profile compared with those from the lower FRT, with IL-1β, IL-6, IL-10, IL-18, CC-chemokine ligand 2 (CCL2; also known as MCP1) and vascular endothelial growth factor (VEGF) levels being markedly higher, and IL-12, IL-15 and MIF levels being markedly lower, in cervical secretions compared with in endometrial secretions 33 . This is probably representative of the unique functions of different FRT compartments — the upper FRT maintains a sterile environment, whereas the lower FRT hosts a population of commensal bacteria. Many of the proteins that are differentially expressed between the upper and the lower FRT and across the menstrual cycle, such as CCL2, IL-6 and IL-1β, are involved in immune cell trafficking and phenotype development. Thus, differences in the levels of specific proteins may account for variations in immune cell populations across the FRT.

At the time of fertilization, during the secretory phase of the menstrual cycle, the FRT must distinguish between a semi-allogeneic fetal placental unit and potential pathogens that are dispersed throughout the FRT during copulation 3 . In preparation for implantation, potential pathogens within the FRT are removed or inactivated, but specific aspects of the innate and adaptive immune responses are regulated to prevent rejection of the fetus. Without these essential conditions being met, successful fertilization, implantation and pregnancy are unlikely to occur. On the basis of our studies and those of others, we proposed that such regulated immune activity results in increased susceptibility to STIs, including HIV 3 . By examining multiple immunological parameters in the lower and upper FRT ( FIG. 5 ), we hypothesized that, during the secretory phase of the menstrual cycle, there is a period lasting 7–10 days that overlaps with the time of implantation, during which important components of innate, humoral and cell-mediated immunity are regulated by OE 2 and P4 in a manner that limits the response to STIs 3 . Various studies have supported the concept of a ‘window’ for HIV infection. Repeated vaginal exposure of pigtail macaques to low doses of simian–human immunodeficiency virus (SHIV) during normal menstrual cycles 122 , 123 showed that the majority of macaques first showed signs of viraemia in the proliferative phase. Taking into account a viral eclipse phase of 7–14 days before viraemia could be detected, these studies estimated a window of most frequent virus transmission between days 24 and 31 of the menstrual cycle (the late secretory phase). In other studies, ex vivo incubation of human cervical explants with HIV showed that productive infection does not occur in tissues from the proliferative phase of the menstrual cycle (which are OE 2 dominated) but only in tissues from the secretory phase 124 . In vitro studies indicate that OE 2 reduces susceptibility to HIV infection in CD4 + T cells and macrophages 125 – 127 . Although experimental models indicate that OE 2 reduces susceptibility to HIV infection, definitive evidence that OE 2 prevents HIV acquisition in women remains to be shown. Several studies indicate that the window of vulnerability may exist for other STIs. For example, gonococcal pelvic infection and chlamydial pelvic inflammatory disease are more likely to occur just before or at the time of menstruation 128 . Mice are most susceptible to N. gonorrhoeae when OE 2 levels are rising, and treatment with OE 2 increases T. vaginalis or C. albicans infection, whereas P4 promotes C. tracho‐matis infection of the lower FRT 129 – 131 . Furthermore, OE 2 increases the attachment of C. trachomatis , T. vaginalis or N. gonorrhoeae to epithelial cells, which is an important factor for establishing infection 132 . OE 2 treatment of ovariectomized mice protects them from HSV2 infection with no demonstrable vaginal pathology or viral shedding 133 . By contrast, P4 treatment of ovariectomized mice makes them highly susceptible to HSV2 infection, with marked pathology, high viral titres in vaginal secretions and persistent inflammation and neutrophil infiltration. This is similar to results observed in non-human primates in which pre-treatment with P4 leads to higher levels of SIV infection 36 . Studies showing different patterns in the hormonal regulation of susceptibility to infection probably reflect variations between animal models, as well as the pathogen target of infection and immune responses to each pathogen. Interestingly, recent studies suggest that immune protection during the proliferative phase of the cycle can be compromised by STI co-infection ( BOX 2 ). Although the exact endocrine conditions responsible for successful infection vary with animal models and cells studied, it is evident that the window of vulnerability provides a useful concept from which to examine the range of pathogens that compromise reproductive health and the lives of women worldwide.

The complexity of immune protection in the FRT requires an understanding of reproductive function and its control by an endocrine system that supports fertilization, implantation and pregnancy. The FRT consists of distinct anatomical sites (Fallopian tubes, uterus, endocervix, ectocervix and vagina) that function separately but in a coordinated manner under the influence of OE 2 and P4. Immune protection throughout the FRT is also precisely regulated by OE 2 and P4. The net result is integrated immune protection that complements the reproductive requirements of each site in the FRT. By examining immune protection in the upper and the lower FRT during the menstrual cycle, a pattern evolves in which aspects of innate, humoral and cellular immunity are either enhanced or suppressed to support both maternal protection and reproductive success. Immune cells, epithelial cells and fibroblasts contribute to a distinct tissue environment in response to OE 2 and P4 that regulates specific immune cell functions throughout the FRT. As a result, the immune conditions that are optimal for fertilization, implantation and pregnancy create a window of vulnerability during the secretory phase of the menstrual cycle, thereby increasing the likelihood of infection by HIV and other STIs. Despite considerable progress in understanding the interface of endocrinology and mucosal immunity in the FRT, much remains to be done to identify the complex mechanisms involved in successful fertility that are proposed to increase the risk of infection by STIs during certain stages of the menstrual cycle. This knowledge will be essential for protecting women from bacterial, fungal and viral pathogens (including HIV) that compromise reproductive health and threaten the lives of women worldwide. Understanding mucosal immune regulation in the FRT will lead to new concepts for therapeutics to enhance tissue and intracellular antimicrobial activity, as well as to the optimization and the development of vaccines and microbicides to prevent sexual transmission of HIV and other STIs to women without compromising reproductive potential.