Microbial infections are a threat to women’s reproductive health. Although reproductive cycles and pregnancy are controlled by sex hormones, the impact of hormones on host–pathogen interactions and immune function in the female reproductive tract are understudied. Furthermore, the changing endocrine environment throughout pregnancy may influence how and when women are susceptible to ascending infection. Because most intrauterine microbial infections originate in the lower reproductive tract, it is vital that future studies determine how different hormonal conditions influence the lower reproductive tract’s susceptibility to infection to understand temporal components of infection susceptibilities across pregnancy. These studies should also extend to nonpregnant women, as it is critical to establish how hormonal fluctuations across the menstrual cycle and hormonal contraceptives may influence disease susceptibility. This review summarizes current knowledge of how estrogen and progesterone impact vaginal and cervical mucosal immunity, barrier function, and interactions with microbial communities.
The female reproductive tract is a unique mucosal environment because of its responsiveness to steroid sex hormones. Relative to other mucosal environments, estrogen and progesterone act through their receptors to induce profound physiological changes to the vaginal, cervical, and uterine mucosa in a cyclical fashion. The mucosal surface of the mammalian lower and upper reproductive tract serves to protect against the outside environment. The lower tract consists of the vagina and ectocervix and is lined with stratified squamous epithelium, which undergoes distinct morphological changes during the estrogen-dominant phase of the estrous and menstrual cycles, when there is a significant increase in epithelial thickness due to proliferation and a decrease in intercellular tight junctions (1). As such, hormonal modulation of the epithelium allows changes in barrier integrity to correspond to physiological requirements for reproduction.
In contrast to the vagina and ectocervix, the endocervical epithelium does not proliferate in response to estrogen; rather, these cells alter their secretory capacity and mucus composition to favor sperm transport (2). During the progesterone-dominant secretory phase of the menstrual and estrous cycles, the vaginal epithelium thins, allowing increased leukocyte trafficking to the vaginal lumen which, in mice, is measurable through vaginal cytology (3). The cervix secretes mucus that contains components that favor protection from pathogens but is less conducive to sperm health and transport (2, 4–8). Thus, the lower reproductive tract is tightly regulated by cyclical fluctuations in estrogen and progesterone. Understanding how these changes impact host–pathogen interactions in the vaginal and cervical mucosal environments is important for improving clinical treatments for sexually transmitted infections and pathogens associated with poor pregnancy outcomes. In this review, we discuss the components of the lower reproductive tract, with particular attention to integration of three biological systems: the immune system, the endocrine system, and the reproductive systems.
The barrier function of the epithelial mucosa of the lower tract is aided by production of mucus from the endocervix, the viscosity of which changes across the cycle. During estrus, for example, the endocervix secretes low-viscosity mucus that lubricates the vaginal lumen to prepare for copulation and optimization of sperm transport (2). Changes in mucus viscosity are achieved by altering the composition of the gel-forming mucins, which are large, negatively charged, highly glycosylated proteins. Four gel-forming mucins comprise human cervicovaginal mucus: MUC2, MUC5AC, MUC5B, and MUC6 (9). Of these, MUC5B is hormonally regulated: in cervicovaginal mucus, its concentrations are highest midcycle (10), suggesting that MUC5B is key to regulating mucus viscosity.
In general, cervicovaginal mucus prevents attachment and colonization of microorganisms to the vaginal epithelium and slows their diffusion, providing opportunity for epithelial and local immune cells to mount a protective inflammatory response when needed. However, some vaginal pathogens have evolved countermeasures to either circumvent or use available gel-forming mucins to persist in the vaginal tract. This includes opportunistic pathogens associated with bacterial vaginosis, a condition in which the vaginal flora is dominated by microbial communities other than Lactobacillus spp., which has been linked to vaginal health. Bacterial species associated with vaginosis, including Gardnerella vaginalis and Prevotella bivia, produce mucinases that degrade the gel-forming mucins, altering the viscosity of the environment (11–14). Similar to commensal organisms, some opportunistic organisms use mucins to establish a niche in the vaginal tract similar to commensal organisms. For example, group B Streptococcus (GBS) upregulates surface pili expression in response to MUC5B, thereby enhancing its ability to bind MUC5B and promote colonization (15). These mechanisms give bacterial pathogens a selective advantage over commensal microbes that depend on mucin binding to colonize and persist in the vaginal tract. Because steroid hormones influence production and composition of gel-forming mucins, it is important to consider how these fluctuations may impact vulnerability to specific pathogens during pregnancy and at different stages of the menstrual cycle.
The cervicovaginal mucus contains other secreted factors, including cytokines, chemokines, and antimicrobial peptides (AMPs) that fluctuate cyclically and may be hormonally regulated (4–8). AMPs such as SLPI, HNP1, HNP2, HNP3, lysozyme, lactoferrin, and surfactant A increase during the proliferative phase and decrease in the secretory phase (16, 17). Estrogen directly suppresses production of the AMPs human β defensin-2 (HBD-2) and elafin by vaginal epithelial cells (17). Critical proinflammatory cytokines such as IL-6, IL-8, and IL-1β, which induce antimicrobial immune responses, also increase during the proliferative phase but fall at ovulation and the luteal phase (16, 18). Thus, the antimicrobial environment of cervicovaginal mucus is hormonally influenced, but further studies are needed to delineate the precise control of specific immune mediators.
Nearly all human and mouse immune cells express estrogen receptors, progesterone receptors, or both (19–21), which permits hormonal immunomodulation and integration of the immune, endocrine, and reproductive systems. Estrogen regulates trafficking and function of T cells, B cells, neutrophils, macrophages, and dendritic cells (DCs) in various tissues, both directly and indirectly, by inducing expression of chemokines and their receptors (19, 21).
Sex hormones modulate the immune function and ecosystem of the reproductive tract across the menstrual and estrus cycles as well as during pregnancy. Great shifts in immune cell numbers and function occur in the uterus across the menstrual and estrus cycles and as pregnancy progresses; however, the lower reproductive tract differs. In rodents, immune cells in the vagina vary across the estrous cycle (22–24), whereas in humans and nonhuman primates, immune cell numbers remain comparatively constant (25–27). However, immune cells in the cervicovaginal fluid or at the transformation zone between the ectocervix and endocervix, where granulocyte populations are largest (26), remain unknown. Furthermore, the function and localization of immune cell populations in the lower reproductive tract during pregnancy are poorly understood.
Estrogen and progesterone are important regulators of neutrophils in various tissues. Estrogen can act indirectly on neutrophils to regulate trafficking by promoting tissue production of chemokines and cytokines, and directly by influencing production of cytokines, microbicidal agents, and free radicals (21, 28–30). Whether estrogen suppresses or enhances neutrophil activity may depend on the context, but several studies suggest that estrogen is proinflammatory, increasing neutrophil extracellular trap formation (NETosis) in human gestation (28), chemotaxis in mammary gland involution (29), and possibly even neutropoiesis (31). In contrast, progesterone has an overall anti-NETotic effect on neutrophils in vitro and reduces production of myeloperoxidase, neutrophil elastase, and TNF-α (28). Collectively, these studies indicate that neutrophil activity is hormonally regulated in various tissues, and the balance between a pro- or anti-inflammatory state is influenced by the overall hormonal milieu, where estrogen tends to promote whereas progesterone dampens neutrophil chemotaxis and activity.
Studies assessing the effects of progesterone and estrogen on neutrophil chemotaxis and activity in the vaginal mucosa tell a different story. For example, in murine proestrus and estrus, when estrogen is high, fewer neutrophils are found in the vagina, whereas in metestrus and diestrus, more neutrophils are present (24, 32). In agreement with this observation, in mice lacking estrogen receptor in the vaginal epithelium, excess neutrophils appear in the vaginal lumen and tissue, regardless of estrus cycle stage (24). This suggests that in contrast to peripheral tissues (24), estrogen inhibits neutrophil trafficking in the vagina.
Estrogen and progesterone also modulate neutrophil adhesion molecules, chemokines, and chemokine receptors in the vaginal tract. CD47 and CD44, which mediate neutrophil transepithelial migration, are inversely regulated by estrogen and progesterone, where estrogen downregulates and progesterone upregulates their expression (24, 33). In mice, both progesterone treatment and genetic deletion of the estrogen receptor promoted neutrophil trafficking into the vaginal lumen and clearance of Candida albicans (33). Interestingly, C. albicans may also be regulated directly by progesterone (34, 35). Likewise, estrogen inhibited and progesterone enhanced production of CXCL1, which drives neutrophil chemotaxis and transepithelial migration into the vaginal lumen (36). Taken together, these studies suggest that estrogen impairs and progesterone promotes vaginal C. albicans clearance through alteration of neutrophil trafficking. No studies have assessed how this impacts neutrophil responses to bacterial pathogens in the vagina or cervix.
Macrophages are broadly categorized into two phenotypes: classically activated M1 and alternatively activated M2 macrophages. M1 macrophages exhibit antimicrobial function through increased chemotaxis, phagocytosis, and production of antimicrobial molecules and proinflammatory cytokines to enhance CD4+ T cell responses (37–40). M2 macrophages are anti-inflammatory and play roles in tissue regeneration, repair, and angiogenesis (41).
Estrogen modulates macrophage function by regulating chemotaxis, cytokine production, phagocytosis, and NO production (21). Estrogen promotes macrophage proliferation and skews them toward an M2 phenotype by dampening IL-1β (42), NO (43), and MMP-9 (43, 44) and increasing IL-4 (42) and IL-10 (45) production. Estrogen can act directly on macrophages through the estrogen receptor (ER)α receptor to inhibit NF-κB nuclear translocation, impairing expression of proinflammatory genes (30, 46–48).
Similar to estrogen, progesterone appears to promote an M2 phenotype. For example, pretreatment of LPS-stimulated macrophages with progesterone reduces NO and IL-12 production in bone marrow–derived and alveolar macrophages (37, 49–51). Progesterone alone decreases phagocytic capacity, oxidative burst, and complement receptor expression in peritoneal macrophages from ovariectomized mice; interestingly, this effect was reversed when cells were cotreated with estrogen and progesterone (52).
Although the effects of sex hormones on macrophage function have been extensively characterized in many tissues, studies assessing how they modulate macrophages in the lower reproductive tract are scarce. Macrophages are sentinels of antimicrobial and immune responses in the vaginal and ectocervical mucosa. In humans, macrophages are abundant in the ectocervix, constituting around a third of the total CD45+ leukocyte population (53), and less so in the vaginal epithelium (26). Compared to the uterus, macrophage numbers remain relatively stable across the menstrual cycle, with only a slight increase during menses (26). However, further studies are needed to increase our understanding of how estrogen and progesterone impact the pathophysiological functions of vaginal and cervical macrophages.
One mechanism by which macrophages control pathogens is by sequestering critical metabolites for their survival, such as iron and manganese. For example, Staphylococcus aureus, GBS, Neisseria gonorrhoeae, Trichomonas vaginalis, C. albicans, and G. vaginalis all acquire iron from their environment for enhanced metabolic function (54–56). Interestingly, M1 and M2 macrophages display differential iron metabolism: M1 macrophages scavenge iron from the extracellular environment, whereas M2 macrophages deposit iron into the extracellular environment (56). Along these lines, estrogen alters iron metabolism by macrophages to increase extracellular iron bioavailability (56, 57), possibly to the detriment of host defense. Further studies are required to assess how estrogen impacts metabolism of iron and other metabolites in the context of lower genital tract infection.
DCs bridge a critical gap between innate and adaptive immunity through their role as professional APCs. During infection, DCs recognize pathogen-associated molecular patterns, process and present Ags, and secrete soluble inflammatory mediators. All of these activities culminate in regulation of T cell immunity. Within the lower reproductive tract, DCs comprise ∼20% of CD45+ immune cells (26, 53), with higher proportions in the ectocervix than in the vaginal epithelium (26, 58, 59). The vaginal mucosa harbors DCs in both the stratified squamous epithelium and in the submucosa (60). Multiple subsets of functionally distinct DCs, including those of the lower reproductive tract, exist in mice and humans (60–63).
Hormonal regulation of DCs occurs in various tissues, including the lower female reproductive tract. Unlike macrophages, estrogen and progesterone have opposing effects on DC differentiation and function. Estrogen stimulates DC maturation and production of proinflammatory cytokines and chemokines (21, 64–66), and it promotes IFN-α production upon activation with viral ligands (67). Furthermore, estrogen enhances TLR-mediated DC maturation, enhancing expression of costimulatory molecules and IL-12 upon exposure to inflammatory stimuli (68). These studies highlight a role for estrogen in priming DC responses to pathogenic stimuli.
Progesterone opposes the effects of estrogen, limiting DC activation and proinflammatory cytokine production, increasing IL-10 production, and reducing stimulation of CD4+ T cells (69, 70). Global gene expression analysis reveals profound metabolic changes in progesterone-treated DCs (71). Furthermore, DCs isolated from mice during diestrus/metestrus are more sensitive to progesterone compared with those isolated during proestrus/estrus, suggesting differential regulation of these cells by sex hormones across the cycle (69). Likewise, circulating human DCs in the third trimester of pregnancy express low levels costimulatory and MHC class II molecules, and they secrete higher IL-10 than IL-12, in comparison with DCs from nonpregnant women (72).
The reduced ability of DCs from progesterone-dominant environments to stimulate T cell activation corresponds with their ability to promote regulatory T cell (Treg) differentiation. Pregnant mice with DCs that lack the progesterone receptor have reduced numbers of CD4+ Tregs and increased intrauterine growth restriction, suggesting that progesterone regulates Treg expansion by modulating DC function during pregnancy (73). Interestingly, high levels of progesterone in pregnancy may negate the proinflammatory effects of estrogen on DCs, even under proinflammatory conditions (74).
How progesterone regulates DC function in the vaginal and cervical mucosa is less clear. Women administered intravaginal progesterone suppositories had triple the number of DCs in the vaginal epithelium compared with untreated women, suggesting that progesterone modulates DC trafficking to the vaginal mucosa (75). Nonetheless, DCs are essential mediators of immune responses to pathogens in the lower reproductive mucosa. DCs efficiently phagocytose and kill C. albicans and stimulate expansion of C. albicans–specific T cells (76, 77). Conversely, DCs isolated from the draining lymph nodes of mice with vulvovaginal candidiasis have low expression of MHC class II and costimulatory molecules, suggesting that C. albicans may tolerize local DCs (78). In a skin model of C. albicans infection, Langerhans cells induced Th17 responses, whereas Langerin+ DCs stimulated Th1 responses (79), highlighting the complex functional dynamics of DC subsets; it is not yet known whether DC subsets in the vaginal mucosa exhibit similar dynamics.
N. gonorrhoeae and C. trachomatis are also important sexually transmitted pathogens that cause significant morbidity among sexually active individuals. N. gonorrhoeae suppresses DC function by inhibiting Th1/Th17 and enhancing Treg expansion (80–82). In contrast, mice treated with DCs pulsed with C. trachomatis were protected against vaginal challenge with the pathogen due to expanded pathogen-specific CD4+ T cell responses (83).
The role of DCs in viral immunity is also extensively studied, including in HIV-1 pathogenesis. In the vaginal mucosa, DCs promote HIV-1 infection and dissemination, serving as a reservoir for HIV-1 replication and persistence (84). In an ex vivo infection model using human vaginal explants, DCs harbored HIV-1, migrated from the site of infection, and passed infectious HIV-1 to neighboring CD4+ T cells (85). Similarly, DCs isolated from human cervix can capture and promote HIV-1 dissemination to CD4+ T cells (58, 59). DCs are also important for HSV-2 immune responses in the vaginal tract: submucosal DCs, but not vaginal LCs, provide protective immunity against HSV-2 infection (86).
Although DCs clearly serve as important players in shaping vaginal and cervical immune responses toward pathogens, we have little information on how steroid hormones impact DC responses to pathogens. Because both progesterone and estrogen strongly influence DC function, future studies should consider their research questions regarding DC immunity to pathogens in the context of various hormonal environments.
Innate lymphoid cells
There are three innate lymphoid cell (ILC) subsets: type 1 ILCs, which consist of type 1 ILCs and NK cells, type 2 ILCs (ILC2s), and type 3 ILCs, which comprise type 3 ILCs and lymphoid tissue inducer (LTi) cells (87, 88). Of the five cell types, ILC2s and NK cells are found in the lower female reproductive tract.
Much of the literature regarding steroid hormone regulation of ILCs is focused on decidual NK (dNK) cells in the uterus due to their specialized role in pregnancy. dNK cells are the predominant leukocyte population in the uterus during pregnancy, comprising 70% in the first trimester (89), and they are phenotypically distinct from conventional NK cells, as they are CD56brightCD16− (90, 91). Functionally, dNK cells differ from conventional NK cells by their decreased cytotoxic capacity (92), secretion of CXCL8 and CXCL10 to promote trophoblast invasion (93), and proangiogenic factors to promote spiral artery remodeling at the site of implantation (94). Both progesterone and estrogen modulate dNK cell migration to the uterus (95) and effector function (96–98). However, one study shows that patient samples isolated during the secretory phase have increased NK cell numbers in the endometrium, but not in the cervix or ectocervix (99), suggesting that progesterone-mediated increases in NK cell chemotaxis may be unique to the uterus. Therefore, given the distinct phenotype and function of dNK cells, further studies are needed to determine whether steroid hormones modulate other ILC subsets in a similar manner.
Conventional NK cells comprise ∼20% of total CD45+ cells in the human ectocervix and cervix and have a greater capacity to produce IFN-γ in response to IL-12 and IL-15 compared with uterine NK cells (100). A humanized mouse model of HSV-2 immunization and vaginal infection shows that human NK cells traffic to the vaginal lumen upon vaginal infection and produce IFN-γ (101). In another study of vaginal HSV-2 infection, NK cells were the predominant sources of IFN-γ early in infection and this is IL-15 mediated, where mice without IL-15 have significantly lower vaginal IFN-γ and succumb to infection (99). These studies demonstrate an important role for NK cells in protection against viral infection in the vagina.
Studies investigating hormonal regulation of NK cells in the lower reproductive tract are scarce. Although NK cells express ERα (102), reports showing estrogen’s direct regulation of NK cell activity have conflicting results, and they only show effects at estrogen concentrations beyond a physiological range (103, 104). One in vivo study shows that ovariectomized mice injected with pregnancy-level estrogen concentrations have reduced peripheral NK cell cytotoxic activity (105), suggesting that estrogen could have an inhibitory effect on NK cell function. However, future studies are needed to confirm this observation and determine the mechanism.
ILC2s are found in the uterus, and similar to dNK cells, their trafficking to this site appears to be hormonally regulated. In ovariectomized mice, ILC2s disappear from uterus; these cells return upon exogenous estrogen treatment. ILC2 abundance in the lungs were unaffected by changing estrogen concentrations (106), suggesting that estrogen modulation is limited to uterine ILC2s. It is possible that this is due to differences in ER expression. ERα expression is low and ERβ and PR expression are absent in ILC2s at various mucosal sites; however, their expression in ILC2s from the female reproductive tract were not measured (107). Moreover, whether estrogen or progesterone regulates ILC2 trafficking in the lower reproductive tract is unknown. One study assessed helminth metabolite induction of type II immunity through ILC2 activation in the vaginal tract and showed that vaginal epithelial cells produce IL-33 in response to helminth metabolites, which in turn stimulated ILC2s to produce the type II cytokine IL-4. Furthermore, this study showed that mice treated with helminth metabolite at estrus produced significantly less IL-33 compared with mice in diestrus, suggesting that the vaginal epithelium is primed to initiate type II responses during periods of high progesterone or low estrogen, possibly through ILC2 stimulation (108). However, the authors did not report whether this also increased IL-4 production from ILC2s and therefore could not conclude whether steroid hormones indirectly impact ILC2 function through IL-33 availability. No other studies have assessed whether progesterone and estrogen modulate ILC2 function in the vagina or cervix.
CD8+ T cells
CD8+ T cells are important mediators of protection against intracellular pathogens, and those found in the endometrium are strongly regulated by hormones. For example, estrogen and TGF-β, but not progesterone, directly suppress cytotoxic activity from endometrial CD8+ T cells. Furthermore, progesterone-treated endometrial epithelial cells secrete TGF-β to levels that suppressed cytotoxic T cell activity (109). In the vagina, CD8+ T cells constitute a substantial percentage of intraepithelial lymphocytes, but unlike uterus, vaginal intraepithelial T cell activity appears to be unaffected by hormone fluctuation (26, 110). Consistent with this, endometrial, but not cervical, T cells from pre- and postmenopausal women show differences in degranulation: endometrial CD8+ T cells of postmenopausal women have increased cytotoxic activity compared with that of premenopausal women. Cervical CD8+ T cells from pre- and postmenopausal women, in contrast, showed similar degranulation (111). These differences may be attributable to inherent differences in vaginal and uterine epithelial and fibroblast responses to hormones, which may subsequently alter local signals impacting CD8+ T cells. However, no such studies have addressed tissue-inherent responses to hormones as they relate to CD8+ T cell activation.
The vaginal mucosa is the primary inductive site for CD8+ T cell responses following exposure to pathogens (112). This can be attributed to the presence of local APCs that prime CD8+ T cells in the vaginal mucosa instead of the draining lymph nodes. Tissue-resident CD8+ memory cells protect against intracellular pathogens in the vaginal mucosa and persist after viral clearance (113–118). For example, HSV-2 reactivation leads to clusters of memory T cells that persist in the vaginal mucosa for months after clearance (118). Whether the induction and maintenance of CD8+ effector and memory T cells are directly influenced by sex hormones in the lower female reproductive tract is unknown. However, the prominent hormonal regulation of DCs strongly suggests that immunity by these cells are at least indirectly affected.
Th1 T cells produce proinflammatory cytokines including IL-2, IFN-y, and IL-12, whereas Th2 cells produce IL-4, IL-10, IL-5, and TGF-β and promote tissue repair. In the uterus and in vitro, estradiol promotes Th1-driven cell-mediated immunity by enhancing IFN-γ production, although at high concentrations it may also stimulate Th2-driven humoral immunity (21). In contrast, progesterone skews CD4+ T cells toward a Th2 profile by suppressing IL-2 and IL-12 production while increasing IL-4 and IL-10 (119). Progesterone induces production of progesterone-induced blocking factor (PIBF), which may promote healthy pregnancy through induction of Th2 type cytokines (120–123). The Th1/Th2 balance is highly correlated with successful in vitro fertilization–embryo transfer delivery: PIBF and IL-4 levels were lower and TNF-α and Th1/Th2 ratios were higher in women who experienced fetal arrest as compared with those who experienced healthy pregnancies (124). These data suggest that the Th1/Th2 balance is critical for implantation and early pregnancy success, and estrogen and progesterone can regulate this balance.
Compared to the upper female reproductive tract, the lower tract has poor Th1 immune responses to pathogens due to high expression of TGF-β and IL-10 (125). During C. trachomatis infection, the lower reproductive tract had higher IL-10 and GATA-3 expression relative to the uterus, suggesting that the vaginal and cervical mucosa have greater presence of Th2 cells. In the same study, Th1 immunity dominated the cervicovaginal mucosa in IL-10–deficient mice, suggesting that this cytokine is critical for maintaining a Th2-skewed immune environment (125). Vaginal biopsies from women with resolved HSV-2 infections show persistence of HSV-2–specific CD4+ memory T cell clusters in the vaginal mucosa, suggesting that they are important for viral clearance and likely protection against future infections. However, the median age of participants in the study was 44–49 y, with menopausal status unreported (118). Regardless, that these immune clusters persisted for months after infection resolution potentially suggests that cyclical hormonal fluctuations do not impact their presence. Furthermore, cyclical hormonal fluctuations do not appear to impact CD4+ T cell numbers in the cervicovaginal mucosa (25). However, whether significant changes in progesterone and estrogen during pregnancy or menopause impact CD4+ T cell abundance in the lower reproductive tract has not been investigated.
Th17 cells are defined by their expression of retinoic acid–related orphan receptor (ROR)γt and the cytokines IL-17, IL-21, and IL-22. Th17 cells play dual roles as mediators of mucosal barrier integrity and homeostasis, as well as protectors against pathogenic infection (126, 127). How progesterone and estrogen impact Th17 abundance and function in the vagina and cervix is poorly understood. However, studies assessing hormonal modulation of Th17 cells in other tissues indicate that these hormones suppress Th17 differentiation and function. For example, progesterone skews CD4+ T cells away from a Th17 phenotype and toward a Th2 phenotype in human and in bovine PBMCs (128, 129). Additionally, progesterone inhibits differentiation of fetal and adult naive T cells into a Th17 cells and promotes Treg differentiation (130). Similarly, in mice, estrus levels of estradiol reduced infection-induced Th17 effector cytokines, albeit indirectly through reduced DC IL-23 production (131, 132), whereas pregnancy levels skew Th17 cells toward a Th2/Treg phenotype (133). Likewise, estrogen-deficient mice have increased circulating Th17 cell numbers and cytokines (134).
Studies in mice suggest that Th17 cells and IL-17 are mediators of protective immunity during vaginal infections. C. albicans clearance correlates with Th17 cells in the vaginal mucosa (135). Additionally, the protective effect of vaccines against C. trachomatis was strongly correlated with their ability to induce Th17 cells (136). Whether Th17 immunity is strongly induced during vaginal infections in women is less clear. Women with C. trachomatis and N. gonorrhoeae have higher levels of IL-17 in cervicovaginal fluid than do uninfected or virally infected women, suggesting that this cytokine is important in bacterial immunity. However, because cytokine levels did not correlate with Th17 cell numbers, IL-17 may be produced by other cell types (137).
It remains unclear whether estrogen and progesterone directly modulate Th17 cells during infections of the lower reproductive tract. In a mouse model of N. gonorrhoeae vaginal infection, estrogen decreased Th17 responses and promoted Treg differentiation, prolonging N. gonorrhoeae persistence (138). In response to C. albicans vaginal infection, Th17 responses were inhibited by estrus-level quantities of estrogen in mice (131). Countering these observations, in a mouse model of HSV-2 infection, mice treated with estrogen after HSV-2 vaccination were better protected against HSV-2 intravaginal challenge than were their untreated counterparts, and this was attributable to high levels of Th17 immunity at the vaginal mucosa (139, 140). These conflicting observations indicate that further studies are needed to better understand how sex hormones impact Th17 responses to vaginal pathogens in both mice and humans.
Th17 cells are a primary target for HIV/SIV at mucosal sites (141–143), and loss of Th17 cells to infection increases viral burden and hastens disease progression (144–146). HIV infection is strongly associated with hormonal fluctuations; acquisition significantly increases during the third trimester of pregnancy, possibly because the target receptor, CCR5, is increased on PBMCs of pregnant women and may be regulated by progesterone (147–149). Furthermore, estrogen may reduce susceptibility of CD4+ T cells to HIV by downregulating CCR5 expression (150). Additionally, cervical explants isolated from women in the secretory (progesterone-dominant) phase were more susceptible to HIV infection than those isolated during the proliferative (estrogen-dominant) phase (151). Likewise, pigtail macaques are more susceptible to simian HIV infection during the secretory phase (152). Collectively, these studies suggest that estrogen and progesterone are important modulators CD4+ T cell susceptibility to HIV.
In the uterus, regulatory Tregs play an essential role in maintaining tolerance throughout pregnancy. In both humans and mice, Treg abundance increases at the maternal–fetal interface and peripherally as pregnancy progresses (153–155), where Treg numbers peak in the second trimester in women (154). Early in pregnancy, Tregs are important for maintaining maternal immune tolerance to fetal cells during implantation (156, 157), and seminal fluid containing paternal Ags has been shown to induce Treg expansion in the uterus to promote tolerance of the conceptus during implantation (156, 158, 159). It follows that aberrant Treg function or abundance can lead to reproductive complications. Women with recurrent spontaneous abortion have decreased numbers of peripheral and decidual Tregs, and suppressive capacity of Tregs in these women is also decreased in comparison with fertile women (160). Furthermore, depleting Tregs in early pregnancy of mice leads to dysfunctional uterine artery remodeling, increased fetal resorption, and a pre-eclamptic phenotype (161). Lower Treg numbers are found at the maternal–fetal interface in women with early onset preeclampsia compared with healthy pregnancies (154). Tregs are also implicated in the pathophysiology of preterm birth, where reduced numbers are found at the maternal–fetal interface in women with idiopathic preterm labor and, in mice, depletion of Tregs in late gestation induces preterm birth (162). Taken together, these studies reveal an important role for Tregs in establishing successful maternal immune tolerance and suppression in the upper reproductive tract to maintain healthy pregnancy outcomes.
Sex hormones regulate Treg abundance and differentiation. Several studies show that estrogen increases abundance and suppressor function of Tregs in various tissues. In mice, estrogen at physiological doses induces Treg expansion in vitro and in vivo (163). In the gut, Treg suppressive function is regulated by estrogen signaling through ERβ: global ERβ knockout mice have reductions in Treg abundance and suppressor function and exacerbated intestinal inflammation (164). Across the menstrual cycle, peripheral Treg numbers are positively correlated with circulating estrogen, where the greatest expansion of CD4+CD25+FOXP3+ Tregs occur late in the follicular phase (165). Furthermore, estrogen therapies reduce symptoms of diseases with dysfunctional Treg etiology, including autoimmune arthritis (166) and autoimmune encephalomyelitis (167). Taken together, these observations demonstrate the positive regulation of estrogen on Treg number and function.
Progesterone similarly enhances Treg abundance; however, this may be species-specific. Ovariectomized mice treated with midpregnancy levels of progesterone had significantly expanded CD4+CD25+ Treg populations compared with mock-treated controls, and in vitro, CD4+CD25− T cells treated with midpregnancy levels of progesterone were converted to CD4+CD25+ Tregs (168). In human cord blood, progesterone promotes the differentiation of fetal T cells into Tregs and suppresses their differentiation into Th17 cells; however, this same effect was not observed in adult peripheral blood Tregs (130), suggesting that progesterone-induced Treg expansion in adults may be specific to mice. Further studies are needed to clarify these discrepant observations.
In the lower reproductive tract, Tregs are important mediators of protection against pathogenic microbes and establishing a tolerogenic niche to commensal microbes and sperm. Treg-depleted mice are unable to mount effective immune responses and clear HSV-2 in the vagina due to reduced DC trafficking to draining lymph nodes to prime effector CD4+ T cell responses (169) and delayed infiltration of NK cells, DCs, and CD4+ T cells to the vaginal mucosa (170). Furthermore, individuals that have high resistance to HIV-1 infection have increased levels of circulating Tregs (171) and increased numbers of endocervical Tregs is associated with lower HIV-1 target cell abundance (172). Furthermore, cord blood from HIV-negative infants born to HIV-1 seropositive mothers have increased Treg numbers and decreased CD4+ and CD8+ cell numbers compared with infants from seronegative mothers. In vitro, Treg depletion from cord blood resulted in increased CD4+CD8+ number and activation (173). These studies suggest that Treg suppressor functions reduce HIV-1 target cell abundance and are thus protective in both the lower reproductive mucosa and the placenta. Tregs protect against Toxoplasma gondii at the maternal–fetal interface through expression of inhibitory molecules CTLA-4 and PD-1 (174); however, the role of Tregs in T. gondii infection at the vaginal or cervical mucosa is unestablished. Tregs may promote infection with some bacterial sexually transmitted infections. Vaginal infection with N. gonorrhoeae increases the number of Tregs in the draining lymph nodes (175) as well as IL-10 production; additionally, blocking Treg and IL-10 activity increased Th1, Th2, and Th17 immunity and promoted clearance of N. gonorrhoeae (81).
There is clear evidence of Treg-mediated immunity in lower reproductive mucosa and hormonal regulation of Treg abundance and function in the upper reproductive tract and periphery. However, whether progesterone and estrogen modulate Treg function in the vaginal and cervical mucosa during infection has not been extensively studied. Future efforts should assess how hormonal fluctuations in pregnancy or across the menstrual cycle influence Treg-mediated immunity to infection in the lower female reproductive tract.
γδ T cells
γδ T cells constitute a small percentage of the total T cell population, but they localize to and exist in greater numbers at mucosal sites. Expression of the γδ TCR endows these cells with a broader range of both innate and adaptive functions than classical αβ T cells (176, 177). γδ T cells are among the first responders in an inflammatory response at mucosal sites, and they rapidly secrete IL-17 upon exposure to pathogens or proinflammatory cytokines released from APCs.
Similar to other immune cell populations, γδ T cells express progesterone and estrogen receptors (178, 179). Hormonal regulation of γδ T cells has primarily been investigated in the context of implantation and spontaneous abortion (179, 180). Progesterone appears to be especially important for regulating γδ T cell trafficking and activity: progesterone concentration is strongly correlated with γδ T cell recruitment to the endometrium and decidua, and abnormal frequencies of γδ T cells are associated with spontaneous abortion (180). Progesterone signaling induces γδ T cell PIBF expression and IL-10 secretion, which in turn promotes proliferation and invasion of trophoblasts (181).
Hormonal regulation of γδ T cells in the lower reproductive tract is not well characterized. γδ T cells are highly abundant in the vaginal and cervical mucosa and are important producers of IL-17 for pathogen clearance, including N. gonorrhoeae (182), C. albicans (183), C. trachomatis, HSV-2 (184, 185), and SIV in nonhuman primates (186). One study reveals γδ T cells as the primary source of IL-17 in the murine reproductive tract under homeostatic conditions, and this was increased upon estrogen treatment (185). Another study shows an association between decreased γδ T cell abundance in the vaginal mucosa and bacterial vaginosis in women (187). Taken together, these studies may point to γδ T cells as mediators of tissue homeostasis in the female genital tract, where disruption leads to commensal microbial dysbiosis. However, further studies are needed to adequately define the role of γδ T cells in vaginal and cervical mucosa and how hormones influence their functions.
B cells comprise a small percentage of the total CD45+ population in both the upper and lower reproductive tract (188). However, their secreted components, Igs, play an important role in protection against pathogens, and Ig concentrations are hormonally influenced (32, 189, 190). Regardless, few studies have assessed how B cell activity or their differentiation to plasma cells is regulated by hormones in the female reproductive tract.
Hormonal regulation of B cells has been extensively studied in the context of autoimmunity and, in particular, systemic lupus erythematosus. These studies have elucidated a clear relationship between systemic estrogen and progesterone levels and B cell population maintenance and function (191). Moreover, multiple studies demonstrate changes in B cell abundance during pregnancy, further implying hormonal regulation. As pregnancy progresses, B cell lymphopoiesis diminishes, which leads to reduced numbers of circulating B cell in the third trimester (176, 192, 193). Contraction of the B cell population is associated with decreased IL-7 levels in the third trimester in mice, and B cell lymphopoiesis can be rescued by exogenous IL-7 administration (192), suggesting that hormonal modulation of B cell population dynamics in pregnancy may be due to alterations in bioavailable IL-7. These observations are unsurprising, given the role of progesterone signaling in thymic involution during pregnancy (194), which is thought to contribute to maternal tolerance through decreased T cell lymphopoiesis. Collectively, these studies implicate steroid sex hormones as regulators of B cell population dynamics and function in pregnancy.
In the lower female reproductive tract, B cells constitute a small percentage of immune cells in the cervix and vagina, and their numbers do not appear to fluctuate across the menstrual cycle. However, Ig levels in cervicovaginal fluid do fluctuate across the estrous and menstrual cycles. Cervicovaginal secretions of IgG are highest during times of increased progesterone and lowest at ovulation in mice (32), humans (189), and nonhuman primates (190). Similarly, estrogen, but not progesterone, decreases Ig receptor expression in the vaginal epithelium, suggesting an overall reduced humoral response capacity around the time of ovulation (22). Thus, although B cell numbers may not fluctuate across the menstrual cycle, shifts in Ig levels suggest that plasma cell activity may be hormonally regulated.
B cell–mediated humoral immunity is important for protection against vaginal pathogens. B-cell-deficient mice exhibit prolonged genital infection with C. trachomatis (195). Furthermore, pathogen-specific neutralizing Abs in the cervicovaginal fluid of mice and women are correlated with protection against GBS vaginal colonization as well as fetal infection during pregnancy (196–199). In mice, vaccination with GBS cell surface proteins induces neutralizing Abs that reduce intrauterine fetal demise and prolong neonatal survival (200). Ab-depleted mice are unable to resolve C. trachomatis genital infection, and passive immunization with convalescent serum from C. trachomatis–infected wild-type dams was protective (201). These studies demonstrate that humoral immunity provides critical protection against some vaginal bacterial pathogens.
Similarly, Igs are important for protection against viral vaginal pathogens. In a mouse model of vaginal HSV infection, IgG is the primary protective component in cervicovaginal mucus, where even nonneutralizing Abs could trap HSV in the presence of mucus and significantly reduce infection (202). These results suggest that mucus and Igs synergize in protection against pathogens. Given the importance of humoral immunity in protection against vaginal pathogens in pregnant and nonpregnant women and how B cell–mediated immunity changes across gestation, it is critical that future studies assess how these facets of humoral immunity contribute to ascending infections during pregnancy, susceptibilities throughout the menstrual cycle, and to determine optimal vaccination practices for pregnant women or women who are planning to become pregnant.
A homeostatic niche within the vaginal and cervical mucosa is maintained by commensal microbes. Commensal flora dominated by Lactobacillus spp. correlates with vaginal health and successful pregnancy outcomes, although not all are dominated by Lactobacillus (203). Bacterial vaginosis occurs when anaerobic bacteria assume dominance, causing chronic community dysbiosis. This poses an increased risk for coinfection with sexually transmitted pathogens, as well as adverse pregnancy outcomes including perinatal infection, miscarriage, and preterm birth.
Lactobacillus spp. employ many mechanisms that inhibit pathogenic competition. They use glycogen deposited by vaginal epithelial cells to produce lactic acid, maintaining a low physiological pH in which pathogens including G. vaginalis and N. gonorrhoeae fail to thrive (204). The lactic acid isomer is also likely important, with d- but not l-lactic acid producing species protecting against C. trachomatis infection (205). Lactic acid and Lactobacillus spp. also regulate the immune environment. Lactic acid induces an anti-inflammatory state in vaginal and cervical epithelial cells (206), and Lactobacillus spp. suppress pathogen-induced immune activation of vaginal epithelial cells by inhibiting proinflammatory cytokines (207).
Lactobacillus abundance in the vaginal mucosa is modulated by steroid sex hormones. Species abundance and diversity fluctuate across the menstrual cycle, where α diversity increases and Lactobacillus abundance decreases during menses, when estrogen declines (208). This observation was confirmed by Krog et al. (209), who also showed that Lactobacillus dominance returns during the luteal and proliferative phases, correlating with increasing serum estradiol. Interestingly, estrogen regulates glycogen production, suggesting that increased bioavailability of glycogen drives Lactobacillus abundance and lactic acid production (210). Elucidating how hormonal fluctuations impact the homeostatic interaction of Lactobacillus and the vaginal mucosa is crucial to understanding susceptibilities to pathogenic microorganisms that compete with lactobacillus in the lower reproductive tract.
Another indicator that sex hormones regulate the vaginal microbiome is evident in pregnancy, which is associated with a stable microbiome, low α diversity, and Lactobacillus dominance (211, 212). Increases in estrogen and progesterone during pregnancy correlate with increases in Lactobacillus, whereas postpartum this shifts to a non–Lactobacillus-dominant community with increased α diversity (212). Hormonal contraceptives also alter the vaginal microbiota and Lactobacillus dominance: estrogen-containing combined oral contraceptive promotes Lactobacillus dominance and decreases α diversity, whereas estrogen-containing vaginal contraceptives and injectable progestin-only contraceptives promoted bacterial vaginosis–dominant community state types and increased α diversity (213). That two estrogen-containing contraceptives had opposite effects on the vaginal microbiota suggests that the presence of estrogen alone does not necessarily increase Lactobacillus dominance. Overall, these studies exhibit the complex dynamics between the endocrine environment and the stability and composition of vaginal microbial communities.
A window of susceptibility to infection in the reproductive tract at specific points during pregnancy and across the menstrual cycle due to hormonal immune modulation has been proposed previously (6). The evidence presented in this review supports the notion that hormones impact immunity in the female reproductive tract, which alters susceptibility to infection (Fig. 1). However, hormonal immune modulation is nuanced and must be carefully and individually considered for each sexually transmitted infection or pathogen that afflicts female reproductive health. For example, mice are most susceptible to N. gonorrhoeae, T. vaginalis, and C. albicans as serum estrogen levels increase. Mouse models of persistent N. gonorrhoeae or GBS vaginal infection require pretreatment with 17β-estradiol (214–216), and in humans N. gonorrhoeae is more often obtained during the proliferative phase (217). In contrast, C. trachomatis is shown to have increased endometrial epithelial attachment with estrogen treatment (218), and yet another study shows that estrogen protects against C. trachomatis persistence while progesterone promotes long-term infection (219). Finally, progesterone and its derivatives have been shown to increase susceptibility to HIV (220), SIV (in nonhuman primates) (221), and HSV-2 (222). These examples highlight complex host–pathogen dynamics influenced by hormones at the female reproductive mucosa. Furthermore, the literature discussed in this review demonstrate clearly that sex hormones have a significant impact on immune dynamics in the lower female reproductive tract (Fig. 1). Thus, it is imperative that future research questions addressing microbial pathogenesis in the lower female reproductive mucosa be considered in the context of the changing sex hormone milieu.
This work was supported by the National Institute of Allergy and Infectious Diseases Grant R21 AI142173, Eunice Kennedy Shriver National Institute of Child Health and Human Development Grant R01HD100832, and National Institute of Allergy and Infectious Diseases Grant R21 154192. Additional support was received through U.S. Department of Agriculture National Institute of Food and Agriculture Grant MICL02447.
The authors have no financial conflicts of interest.