The Role of Sex Hormones in Immune Protection of the Female Reproductive Tract

The role of sex hormones in immune protection of the female reproductive tract Charles R Wira Marta RodriguezGarcia and Mickey V Patel Department of Physiology and Neurobiology Geisel School of Medicine at Dartmouth Lebanon New Hampshire 03756 USA Abstract Within the human female reproductive tract FRT the challenge of protection against sexually transmitted infections STIs is coupled with the need to enable successful reproduction Oestradiol and progesterone which are secreted during the menstrual cycle affect epithelial cells fibroblasts and immune cells in the FRT to modify their functions and hence the individuals susceptibility to STIs in ways that are unique to specific sites in the FRT The innate and adaptive immune systems are under hormonal control and immune protection in the FRT varies with the phase of the menstrual cycle Immune protection is dampened during the secretory phase of the cycle to optimize conditions for fertilization and pregnancy which creates a window of vulnerability during which potential pathogens can enter and infect the FRT Many challenges including gynaecological cancers and sexually transmitted infections STIs threaten reproductive health by escaping the protection that is conferred by the mucosal immune system In 2012 the worldwide incidences of ovarian cancer 239000 cases uterine cancer 320000 cases and cervical cancer 528000 cases the last of which is primarily caused by human papillomavirus HPV infection were among the highest of all lifethreatening diseases see World Cancer Research Fund International Data on Specific Cancers The World Health Organization WHO estimates that in 2008 there were at least 498 million new cases of the more than 30 known STIs including infection with Trichomonas vaginalis 276 million new cases Chlamydia trachomatis 106 million new cases Treponema pallidum 10 million new cases HIV 27 million new cases and Neisseria gonorrhoeae 106 million new cases all of these infections can lead to reproductive failure and death1 Women are at a greater risk of STIs than men Prevalence rates and total case numbers for C trachomatis N gonorrhoeae and T vaginalis infection are higher in women than in men2 In SubSaharan Africa women account for two out of three new infections with HIV and in the United States genital herpes infects one in five women compared with one in ten men see Genital Herpes CDC Fact Sheet Despite our growing understanding of the mucosal immune system in the female reproductive tract FRT much remains to be learnt about the underlying mechanisms that regulate susceptibility to STIs in the FRT Correspondence to CRW CharlesRWiraDartmouthedu Competing interests statement The authors declare no competing interests HHS Public Access Author manuscript Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Published in final edited form as Nat Rev Immunol 2015 April 154 217230 doi101038nri3819 Author Manuscript Author Manuscript Author Manuscript Author Manuscript The mucosal immune system is the first line of defence against a complex range of viral bacterial fungal and parasitic pathogens In common with other mucosal sites the innate and adaptive both cellular and humoral elements of the mucosal immune system have evolved to meet the special challenges that are associated with the FRT Unique among mucosal sites the FRT has evolved to accept a semiallogeneic fetus and to confer protection against potential pathogens Important to this balance is the regulation of the FRT immune system by the sex hormones oestradiol OE2 and progesterone P4 The FRT can be divided into a lower tract vagina and ectocervix and an upper tract endocervix uterus and Fallopian tubes FIG 1 Each compartment has distinct reproductive responsibilities sperm entry ovum movement nutrition or preparation for implantation that coincide with distinct phases of the menstrual cycle Sex hormones coordinate unique patterns of epithelial cell stromal fibroblast and immune cell function which optimize conditions for both maternal protection and fetal survival This Review focuses on current knowledge regarding the sentinel role of the mucosal immune system in the FRT with a special emphasis on the interface between the immune system and the endocrine system We describe the immune changes that occur in vivo during the menstrual cycle as well as those that occur in vitro after treatment with sex hormones As a result of the complexity of immune regulation in the human FRT it is beyond the scope of this Review to examine the immune changes that occur during adolescence pregnancy or menopause or that are associated with sexual assault or gynaecological disorders In the following sections we define the changes in hormone levels that occur during the menstrual cycle identify the cells responsible for innate and adaptive immune protection in the reproductive tract and focus on the role of sex hormones particularly OE2 and P4 in regulating epithelial fibroblast and immune cell phenotype and function Special emphasis is given to our limited but growing knowledge of the sitespecific immune responses in the upper and lower FRT and how each cell type contributes through the secretion of growth factors cytokines and chemokines to a tissue environment that maintains immune protection and reproductive potential Finally we discuss the concept of a window of vulnerability in the menstrual cycle during which immune regulation as a result of changes in hormone levels optimizes conditions for fertilization and implantation but places women at increased risk of acquiring STIs3 Endocrine control of the menstrual cycle The hypothalamicpituitary axis regulates the cyclic secretion of OE2 and P4 by the ovary during the menstrual cycle in women of reproductive age In response to these hormones changes take place throughout the reproductive tract in preparation for egg production potential fertilization implantation in the uterus and pregnancy The menstrual cycle is divided into four stages the menstrual phase the proliferative phase also known as the follicular phase midcycle during which ovulation occurs and the secretory phase also known as the luteal phase BOX 1 FIG 2 The length of a menstrual cycle varies greatly among women 2135 days with 28 days used as the average length At the beginning of the menstrual cycle day 0 folliclestimulating hormone FSH stimulates ovarian thecal and granulosa cells in the developing follicle to produce OE2 Under the control of the hypothalamicpituitary axis through FSH and luteinizing hormone OE2 levels Wira et al Page 2 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript increase during the proliferative phase to peak just before ovulation occurs at midcycle This is followed by a surge in luteinizing hormone that is essential for ovulation which occurs 2436 hours after OE2 levels peak in the blood Following ovulation the corpus luteum develops to become the primary source of OE2 and P4 during the secretory phase The concentrations of P4 and to a lesser extent OE2 increase and peak at the midsecretory phase In the absence of fertilization the corpus luteum degrades in a process known as luteolysis resulting in a decrease in OE2 and P4 levels which leads to endometrial shedding and the onset of menses4 As discussed below these changes in hormone levels have a marked effect on the immune system in the FRT and we propose that they lead to a window of vulnerability during which optimal conditions for fertilization and implantation increase an individuals susceptibility to STIs FIG 2 Immune protection of the FRT Cell types 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 Multilayered squamous epithelial cells cover the lower FRT vagina and ectocervix whereas singlelayer 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 620 of total cells in the human FRT with more leukocytes being present in the upper tract than in the lower portions of the tract5 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 Patternrecognition receptors PRRs including Tolllike receptors TLRs RIGIlike receptors RLRs and NODlike receptors NLRs are essential for the initial detection and response to pathogens as they recognize conserved pathogenassociated molecular patterns PAMPs For example TLR7 and RIGI recognize HIV whereas TLR2 and TLR4 recognize C trachomatis and TLR2 recognizes N gonorrhoeae611 PRR expression varies within the FRT12 Expression of the bacterial receptors TLR2 TLR4 nucleotidebinding 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 pathogens1314 A similar trend is seen for the cytoplasmic PRRs RIGI and melanoma differentiation associated protein 5 MDA5 also known as IFIH114 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 FRT15 Wira et al Page 3 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 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 phase1619 OE2 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 PRRs20 P4 increases TLR4 expression by fibroblasts which suggests that these cells are more sensitive to bacterial pathogens in the secretory phase17 However it has not been directly shown that the levels of PRR expression correlate with protection against pathogens in the FRT OE2 also modulates the signalling pathways downstream of PRRs and proinflammatory receptors It inhibits the lipopolysaccharide LPS and polyinosinicpolycytidylic acid polyICinduced secretion of macrophage migration inhibitory factor MIF interleukin6 IL6 and IL8 by uterine epithelial cells and reverses the stimulatory effects of IL1β on mRNA and protein expression of tumour necrosis factor TNF human β defensin 2 HBD2 IL8 and nuclear factorκB NFκB This suggests that inflammatory responses to pathogens are decreased during periods of high OE2 levels in the menstrual cycle2122 OE2 regulates the function of NFκB which is a key transcription factor involved in inflammatory gene expression by restricting its cytoplasmictonuclear translocation or by preventing the degradation of NFκB inhibitors2324 Furthermore as secretory leukocyte protease inhibitor SLPI also known as antileukoproteinase inhibits NFκB expression OE2mediated inhibition of proinflammatory cytokine expression may be mediated through the regulation of NFκB by OE2induced SLPI25 Thus OE2 may reduce susceptibility to HIV infection in the FRT by creating an antiinflammatory 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 immuneactivated environment that is often associated with infections Secreted molecules 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 cervicovaginal 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 midcycle day 13 and remain low for 710 days during the secretory phase before returning to the higher levels found during the proliferative phase following menstruation2627 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 factorβ 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 midcycle possibly as a result of cycle length variation BOX 1 and differences in sampling technique2832 By contrast IL6 and IL1β levels increase during the proliferative phase of Wira et al Page 4 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript the cycle which shows that concentration changes are specific to certain molecules at specific phases of the cycle29 Secretions from the upper FRT have a distinct proteomic profile compared with those from the lower FRT with IL1β IL6 IL10 IL18 CCchemokine ligand 2 CCL2 also known as MCP1 and vascular endothelial growth factor VEGF levels being markedly higher and IL12 IL15 and MIF levels being markedly lower in cervical secretions compared with in endometrial secretions33 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 IL6 and IL1β 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 Endocrine control of epithelial cells Barrier function Epithelial cells provide a protective barrier in the FRT that is responsive to hormonal and pathogenic stimuli OE2 increases the proliferation of epithelial cells in both the uterus and the vagina3435 High levels of P4 are associated with thinning of the vaginal epithelium in animal models although this has not been observed in humans36 Epithelial cells are linked by tight junction proteins which regulate the movement of molecules across the epithelium In the stratified squamous epithelium of the lower FRT tight junctions are primarily present between basal epithelial cells37 FIG 1 Their absence in the superficial epithelium results in weakly joined cells and may allow pathogens such as HIV to penetrate the epithelial layer bringing them into proximity with immune cells in the basal epithelium and in the lamina propria By contrast the columnar epithelium in the upper FRT has strong networks of tight junctions OE2 modulates the expression of claudin and occludin proteins which leads to a relaxation of tight junctions and greater flux across the epithelium3839 The functional implications of this are unclear but may involve the movement of proteins across the epithelium as part of normal homeostasis in preparation for potential implantation or as part of the clearance of pathogens in immune defence Whether alterations in barrier permeability throughout the menstrual cycle are associated with the increased movement of pathogens into the subepithelial tissue is unknown However pathogens and inflammatory conditions degrade tight junction integrity and thus barrier function which leads to greater flux across the epithelium40 Therefore a combination of high OE2 levels and inflammation may degrade barrier function in the upper FRT and may increase susceptibility to infection Mucus production Endocervical epithelial cells secrete negatively charged highmolecularweight glycoproteins known as mucins which are a major component of mucus and which trap pathogens and prevent their access to the epithelium Mucin gene expression varies with menstrual status which leads to changes in the overall properties of mucus4142 Oestrogenic mucus is thin and watery with a low viscosity which facilitates sperm movement into the Wira et al Page 5 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript upper FRT It is present during the proliferative phase and increases at ovulation By contrast progestational mucus is thick viscous and present following ovulation and during the secretory phase when it functions to impede the movement of material from the lower FRT into the upper FRT43 Mucus protects epithelial cells from direct contact with pathogens such as HIV Cervicovaginal mucus has recently been shown to interact with IgG antibodies to impede HIV mobility and thereby to enhance mucosal barrier function but it is not known whether this property changes during the menstrual cycle4445 Cytokines chemokines and antimicrobial proteins Epithelial cell secretion of cytokines chemokines and antimicrobial proteins varies with location in the FRT and hormone exposure46 For example OE2 but not P4 suppresses the secretion of the antimicrobial proteins HBD2 and elafin also known as peptidase inhibitor 3 by vaginal squamous epithelial cells3247 By contrast OE2 increases uterine epithelial cell secretion of SLPI and HBD2 with preferential secretion towards the lumen from where incoming pathogens would meet the mucosal surface22 This may enable the FRT to host commensal bacteria in the lower tract and to maintain protection against infection in the upper tract despite being exposed to potential pathogens throughout the menstrual cycle148 In addition capacitated human sperm show directional movement towards CCL20 and HBD2 in chemoattractant assays as a result of their expression of CCchemokine receptor 6 CCR648 The fact that uterine but not vaginal epithelial cells secrete CCL20 at a time when HBD2 is suppressed in the lower FRT provides a gradient that promotes sperm chemotaxis under noninflammatory conditions towards the upper FRT in which fertilization occurs Studies using vaginal epithelial cell lines show that OE2 inhibits the expression of mRNA encoding the inflammatory proteins IL1α and TNF which suggests that inflammation and the resulting influx of immune cells to the epithelial surface are repressed before ovulation at the time that is most amenable for semen entry20 In other studies apical secretions from uterine epithelial cells from premenopausal women but not from postmenopausal women had antibacterial activity against both Grampositive and Gramnegative bacteria and this was dependent on SLPI49 This may lead to decreased protection against bacterial pathogens in postmenopausal women as a result of the absence of OE2 Type I interferons IFNs mediate the antiviral response through their regulation of IFN stimulated genes ISGs In the FRT OE2 regulates the expression of IFNɛ across the menstrual cycle but not of other type I IFNs such as IFNα and IFNβ5051 Furthermore whereas ISG expression by uterine epithelial cells in response to IFNβ is not affected by OE2 OE2 does reduce ISG levels in response to type III IFNs IFNλ1 IFNλ2 and IFN λ350 Together these studies show the specificity of hormone action on epithelial cells and genes in the FRT demonstrating their crucial role in homeostasis and antiviral defence in the FRT Endocrine control of stromal fibroblasts Fibroblasts are essential structural components of the FRT but their role in immune protection is poorly understood Studies have shown that fibroblasts from other mucosal Wira et al Page 6 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript surfaces are involved both in the recognition of pathogens and in the recruitment of immune cells to sites of infection52 Similarly to epithelial cells fibroblasts have sitespecific differences in their responses to sex hormones53 For example during the secretory phase of the menstrual cycle under the influence of P4 and independently of the presence of a blastocyst in the uterine cavity uterine fibroblasts undergo marked phenotypic changes known as decidualization in preparation for implantation This response is not observed in fibroblasts from the cervix and the Fallopian tubes OE2 increases the secretion of hepatocyte growth factor HGF and CXCchemokine ligand 12 CXCL12 also known as SDF1α and decreases CCL2 secretion from uterine fibroblasts but not from fibroblasts of the endocervix or ectocervix which may account for differential recruitment of immune cells into the endometrium at different phases of the menstrual cycle5354 Although both vaginal and uterine fibroblasts proliferate in response to OE2 treatment in vitro vaginal fibroblasts respond at OE2 concentrations that are approximately 1000fold lower than those required for uterine fibroblasts to respond which suggests that vaginal fibroblasts are more sensitive to the presence of OE2 REF 55 Fibroblasts can respond to pathogens that have breached the epithelial barrier by alerting immune cells and recruiting them to sites of infection For example uterine fibroblasts respond to the TLR3 and TLR4 ligands polyIC and LPS by secreting cytokines such as TNF IL8 CCL2 CCL5 also known as RANTES CCL20 and HGF OE2 potentiates HGF secretion by fibroblasts in response to polyIC which suggests that the intensity of the fibroblast immune response to viral pathogens varies with the stage of the menstrual cycle53 Secretions from uterine fibroblasts inhibit CCR5tropic HIV infection of TZMbl cells whereas OE2 pretreatment increases antiviral activity against CXCchemokine receptor 4 CXCR4tropic HIV which shows the potential importance of endocrine regulation of fibroblasts in protecting HIVtarget cells in the FRT54 Endocrine control of immune cells Immune cells in the FRT are regulated by sex hormones throughout the menstrual cycle to maintain the equilibrium between effectively fighting infection and the immune regulation and tissue remodelling that is required for successful implantation and pregnancy3 Immune cell number distribution and function are tightly modulated throughout the menstrual cycle to achieve these goals FIG 3 The result is the migration and differentiation of unique immune cell phenotypes throughout the FRT which are different from those of immune cells at other mucosal sites in the body and in peripheral blood Cell numbers tissue distribution and trafficking The proliferative phase of the menstrual cycle is characterized by the regeneration of the endometrial tissue During this period which is dominated by OE2 angiogenesis occurs as well as glandular epithelial cell and stromal fibroblast growth56 On the basis of multiple studies immune cell numbers in the endometrium are known to increase during the late secretory phase and during menstruation55658 By contrast in the lower FRT sex hormone fluctuations do not alter immune cell numbers which remain constant throughout the cycle59 Around the time of ovulation the peak in angiogenesis facilitates recruitment into the uterus of leukocytes including NK cells neutrophils and macrophages which are Wira et al Page 7 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript necessary should pregnancy occur Cell recruitment is mediated through the cytokines chemokines and growth factors that accumulate in the vicinity of the uterine blood vessels such as CCL4 also known as MIP1β CCL14 CCL16 and CCL21 which are mainly produced by epithelial and stromal fibroblasts under the influence of sex hormones56 T cells constitute around 4050 of leukocytes in the FRT56063 During the proliferative phase most T cells in the uterus are found as scattered T cells and small aggregates in the stroma or as intraepithelial lymphocytes6465 During this time uterine CD8 T cell numbers remain constant but undergo a uterine sitespecific condensation in the lamina basalis which results in the formation of lymphoid aggregates Lymphoid aggregates consist of a B cell core surrounded by memory CD8 T cells and encapsulated by macrophages they peak in size at midcycle and persist during the secretory phase64 In the absence of infection lymphoid aggregates are found in the endometrium but not in the endocervix or lower FRT Aggregate formation may be a mechanism to maintain the T cell repertoire and to prevent the loss of resident memory T cells during menstrual shedding In the lower FRT clusters of cells form in the vagina and cervix in response to herpes simplex virus 2 HSV2 infection66 These clusters contain memory CD4 or CD8 T cells B cells dendritic cells DCs and macrophages and may persist for months or years after viral clearance Whether they are regulated by sex hormones is unknown These cell clusters probably provide protection against secondary infections but at the same time may be a locus of increased susceptibility for other infections such as HIV6667 Macrophages represent about 1020 of the FRT leukocytes56364 In the uterine endometrium CD68 macrophages are found directly below the luminal epithelium and in the subepithelial stroma as well as in clusters in the lamina basalis adjacent to the glandular epithelium65 Macrophages are more abundant in the endometrial stroma68 than in the endocervix or ectocervix and their numbers remain stable during the proliferative phase69 In the upper FRT CD1a and CD11c DCs are located within the luminal epithelium and CD123 plasmacytoid DCs are present in the stroma65 The functionalis layer and basalis layer of the endometrium contain CD1a DCs and fewer numbers of CD83 mature DCs Whereas numbers of CD1a DCs remain constant and are similar in both layers during the proliferative phase CD83 DCs are more abundant in the basal layer70 In the lower FRT DCs are found mostly within the epithelium71 Uterine NK cells which represent approximately 30 of leukocytes during the implantation window increase in number during the secretory phase accompanying decidualization of the endometrium567273 It is unclear whether uterine NK cell numbers increase as a result of the selective recruitment of CD56hiCD16 NK cells from peripheral blood or as a result of in situ proliferation567273 Chemokines and cytokines such as CXCL10 CXCL11 REF 74 and IL15 the levels of which are regulated by sex hormones selectively recruit NK cells In addition IL15 can locally increase uterine NK cell proliferation56 P4 withdrawal initiates menstruation which triggers an inflammatory response in the endometrium58 Chemokine cytokine and growth factor secretion by the endometrial epithelium and stroma regulates the influx of leukocytes that mediate tissue breakdown and Wira et al Page 8 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript repair Neutrophils NK cells macrophages and smaller numbers of eosinophils and CD1a DCs migrate into the uterus in response to hormonal changes5870 The number of CD68 macrophages is increased during menstruation particularly in the midmenstrual phase days 34 decreases towards the end of menstruation and remains stable throughout the proliferative phase69 Immune function and phenotype Successful implantation is associated with immune cell regulation in which an inflammatory response that attracts innate immune cell subsets specialized in tissue remodelling is integrated within a tolerogenic environment that prevents T cellmediated allograft rejection7576 Immune function is hormonally controlled in a sitespecific manner through growth factors cytokines and chemokines that are present in the local tissue environment In the lower FRT CD4 and CD8 T cells are equally abundant whereas in the endometrium CD8 T cells predominate6061 The increased presence of CD4 T cells in the lower tract suggests greater susceptibility to HIV infection at this site In addition CD8 cytotoxic T lymphocyte CTL activity is suppressed during the secretory phase of the cycle in the endometrium presumably to minimize the recognition and the rejection of allogeneic sperm and the semiallogeneic fetus By contrast CTL activity is maintained in the lower FRT offering constant protection against potential incoming pathogens77 Interestingly lymphoid aggregate formation correlates with the loss of CTL activity in that lymphoid aggregates reach maximal size during the secretory phase of the cycle when CTL activity is suppressed6477 These cell aggregates might therefore be a mechanism to prevent T cell mediated rejection of the semiallogeneic fetus Regulatory T TReg cell subsets are also hormonally regulated In the endometrium forkhead box P3 FOXP3 TReg cell numbers increase throughout the proliferative phase and then decrease at the beginning of the secretory phase78 In peripheral blood the number of CD4CD25FOXP3 TReg cells follows the same pattern during the menstrual cycle79 Interestingly the decrease in TReg cell number during the secretory phase does not occur in pathological circumstances such as in recurrent spontaneous abortions or in endometriosis which indicates that the increased number of TReg cells before ovulation may be necessary to induce immune tolerance for successful implantation and for tissue breakdown and repair7879 Furthermore these findings indicate that CTL suppression during the secretory phase is not mediated by TReg cells We recently reported a decreased number of CD4 T helper 17 TH17 cells in the endometrium compared with the cervix from premenopausal women61 TH17 cells are involved in host defence against extracellular bacteria and fungi and their increased number in peripheral blood has been linked to recurrent pregnancy loss80 Experiments using ovariectomized mice show that OE2 deficiency induces TH17 cell differentiation81 Furthermore mouse models have shown the induction of TH17 cell responses by sperm antigens and that this response is inhibited by OE2 at oestrus82 Although variations in TH17 cell number in the FRT throughout the menstrual cycle were not addressed in our study61 we found decreased numbers of TH17 cells in the endometrium from premenopausal women compared with postmenopausal women Decreasing the number of Wira et al Page 9 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript TH17 cells in the endometrium which is possibly mediated by sex hormones may be necessary for successful fertilization and implantation whereas higher TH17 cell numbers are required in the lower FRT to prevent bacterial and fungal infections As TH17 cells are susceptible to HIV infection their presence places the lower FRT at greater risk of HIV infection than the endometrium Whereas B cells are a minor cell population in all FRT tissues IgG and IgAproducing plasma cells are predominantly found in the cervix and to a lesser extent the vagina83 In FRT secretions IgG is partly locally produced and partly derived from the circulation Cervicovaginal secretions are characterized by greater amounts of IgG than IgA Interestingly in both humans and rodents uterine and cervicovaginal levels of IgA and IgG are hormonally regulated83 Despite the low numbers of IgAproducing plasma cells in the endometrium8486 levels of stromal IgA and IgG increase during ovulation8788 By contrast in cervical secretions both IgG and IgA levels are lowest at the midsecretory phase of the menstrual cycle8990 Suppression of IgG and IgA levels at midcycle in the lower FRT is thought to reduce the levels of spermspecific antibodies which would otherwise contribute to infertility As discussed elsewhere immunoglobulin changes during the menstrual cycle at each site are probably the result of endocrine regulation of receptors for IgA the polymeric IgA receptor pIgR and IgG the neonatal Fc receptor FcRn in epithelial cells of plasma cell synthesis and of transudation of immunoglobulins from blood into FRT tissues9192 In humans pIgR production by uterine epithelial cells varies with the stage of the menstrual cycle with the greatest quantities being produced during the secretory phase93 By contrast pIgR production in vaginal epithelium is minimal94 In rodents OE2 stimulates the production of secretory component the external domain of pIgR by uterine epithelial cells but inhibits production by vaginal epithelial cells95 Overall these findings suggest that antibodies secreted into the upper tract contribute to the removal of potential pathogens by inhibiting cell entry andor by neutralizing the biological activity of a pathogen Antibody binding further mediates pathogen removal through phagocytosis by macrophages or through the complement system The lack of immunoglobulinproducing cells in the upper FRT suggests that antibodies of the upper tract are less likely to be of local origin and that they are possibly derived from the gut Evidence for a gut origin comes from studies of IgA cell traffic from the gut via the mesenteric lymph nodes to the blood and into mucosal sites9699 Interestingly differences between species have been reported for example immunoglobulinsecreting plasma cells are found in the mouse uterus but not in the human uterus8486100 An explanation for this difference is that whereas semen is deposited into the vagina of humans it is placed directly into the uterus of mice where it possibly elicits a more pronounced inflammatory response than that seen in the human uterus reviewed in REF 92 NK cells in the FRT express CD9 and have distinct sitespecific phenotypes73 Ectocervical and vaginal NK cells are CD56CD16 but lack expression of CD94 and CD69 which is similar to the phenotype of CD56lowCD16 cytotoxic NK cells in the blood7273 By contrast NK cells of the upper FRT are CD56CD16 CD94CD69 and express the activating receptors natural killer group 2 member D NKG2D and NKp30 also known as NCR3 but not NKp44 also known as NCR2 or NKp46 also known as NCR1 which differentiates these cells from the decidual NK cells that are found during pregnancy72 Wira et al Page 10 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Endometrial NK cells have low levels of cytotoxic activity cytokine secretion and pro angiogenic factor production Decidual NK cells and endometrial NK cells are different cell subsets and it is unclear whether endometrial NK cells have tissueremodelling functions similarly to decidual NK cells or whether they are inactive cells awaiting pregnancy72101102 Intracellular expression of IFNγ by uterine NK cells is suppressed by epithelial cell production of TGFβ103 and NK cell cytotoxicity and perforin production are inhibited by P4 REF 58 Thus the cytolytic activity of CD8 T cells and of uterine NK cells is suppressed in the endometrium during the secretory phase of the menstrual cycle Additional innate lymphoid cell ILC subsets other than NK cells have recently been described in human decidua but their presence in the FRT of nonpregnant subjects has not been investigated104 Considering the central role of ILCs in tissue remodelling antimicrobial responses and maintenance of epithelial barrier integrity reviewed in REF 105 their characterization function and hormonal regulation in the FRT is an important area for future studies Further indication of immune cells adopting a specialized cell phenotype in the endometrium comes from the high proportion of CD163CD14low macrophages which are known as alternatively activated macrophages106 These cells are distinct from those in the cervix and vagina which express high levels of CD14 REFS 63107 Macrophages sustain HIV infection in the lower and upper tract107108 By contrast CD4 T cells in the endometrium are poorly susceptible to HIV infection61 which suggests that macrophages may be the main HIVtarget cell in the upper FRT rather than CD4 T cells Endocrine control of the mucosal environment It is important to view the immune system of the FRT not as a set of isolated cell types but rather as part of a mutually interdependent network The multidirectional interactions between epithelial cells fibroblasts and immune cells are essential for maintaining reproductive health and immune protection Epithelial cell interactions with underlying stromal fibroblasts and immune cells are essential in facilitating sex hormoneinduced changes FIG 4 Epithelial cells contribute to the tissue environment by basolaterally secreting a range of growth factors and cytokines in response to OE2 including TGFβ granulocytemacrophage colonystimulating factor GMCSF macrophage colony stimulating factor MCSF IL4 IL6 TNF IL8 and IL10 REFS 109111 Uterine fibroblasts respond to OE2 by secreting paracrine factors such as HGF that mediate hormone effects on epithelial cell growth and differentiation as well as by increasing blood supply to the endometrium112113 Fibroblasts throughout the FRT secrete CCL2 and IL8 and there is some evidence that their secretion is partially controlled by OE2 REFS 114115 Cytokines and chemokines produced by immune cells such as TNF CCL5 fibroblast growth factor 2 FGF2 and GMCSF also affect other immune cells as well as fibroblasts and epithelial cells106 These findings indicate that epithelial cells fibroblasts and immune cells in the FRT are under hormonal control to create an optimal tissue environment at the time of implantation the secretory phase of the cycle to enable successful reproduction Wira et al Page 11 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript At the same time secretions in the tissue environment function to modulate immune protection For example epithelial cell secretions have marked effects on immune cell phenotype such as conferring a more tolerogenic phenotype on DCs116117 TGFβ is a particularly potent immunomodulator that is responsible for the downregulation of expression of DCspecific intercellular adhesion molecule 3grabbing nonintegrin DC SIGN also known as CD209 on immature DCs which reduces HIV transinfection to target cells116117 CCL20 directly inactivates HIV but also attracts CCR6 cells to the mucosal surface some of which are uniquely susceptible to HIV infection118119 For example CD4CCR6 T cells express the HIV coreceptor CCR5 and have increased susceptibility to in vitro HIV infection compared with CD4CCR6 T cells61 Thus CCL20 can function as both an inhibitor and a promoter of HIV infection Other antimicrobial proteins can also modulate intracellular cell signalling pathways in addition to their HIVspecific effects For example elafin and SLPI secretion of which by uterine epithelial cells is increased by OE2 dampen the inflammatory response to TLR3 and TLR4 ligands by decreasing TLR expression andor by reducing NFκB activation and nuclear translocation25120 HBD2 another antimicrobial factor that is regulated by OE2 functions as a ligand for TLR4 in immature DCs leading to DC maturation121 Thus OE2 can potentially increase antiviral activity in secretions can induce signalling in immune cells and can alter the response to PRR ligands as well as regulate the influx of immune cells through a restrained inflammatory response Endocrine control of a window of vulnerability At the time of fertilization during the secretory phase of the menstrual cycle the FRT must distinguish between a semiallogeneic fetal placental unit and potential pathogens that are dispersed throughout the FRT during copulation3 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 HIV3 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 710 days that overlaps with the time of implantation during which important components of innate humoral and cellmediated immunity are regulated by OE2 and P4 in a manner that limits the response to STIs3 Various studies have supported the concept of a window for HIV infection Repeated vaginal exposure of pigtail macaques to low doses of simianhuman immunodeficiency virus SHIV during normal menstrual cycles122123 showed that the majority of macaques first showed signs of viraemia in the proliferative phase Taking into account a viral eclipse phase of 714 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 OE2 dominated but only in tissues from the secretory phase124 Wira et al Page 12 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript In vitro studies indicate that OE2 reduces susceptibility to HIV infection in CD4 T cells and macrophages125127 Although experimental models indicate that OE2 reduces susceptibility to HIV infection definitive evidence that OE2 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 menstruation128 Mice are most susceptible to N gonorrhoeae when OE2 levels are rising and treatment with OE2 increases T vaginalis or C albicans infection whereas P4 promotes C trachomatis infection of the lower FRT129131 Furthermore OE2 increases the attachment of C trachomatis T vaginalis or N gonorrhoeae to epithelial cells which is an important factor for establishing infection132 OE2 treatment of ovariectomized mice protects them from HSV2 infection with no demonstrable vaginal pathology or viral shedding133 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 nonhuman primates in which pretreatment with P4 leads to higher levels of SIV infection36 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 coinfection 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 Conclusions 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 OE2 and P4 Immune protection throughout the FRT is also precisely regulated by OE2 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 OE2 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 Wira et al Page 13 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 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 Acknowledgments The authors express their appreciation to J Fahey for help in editing this manuscript This work was supported by US National Institutes of Health NIH grants AI102838 AI071761 and AI117739 Glossary Implantation The binding to and invasion of the uterine endometrium by the blastocyst which occurs 512 days after fertilization Proliferative phase Days 514 of the classical menstrual cycle Defined as the period between the end of menstrual bleeding and ovulation Characterized by rising serum levels of oestradiol and very low levels of progesterone Secretory phase Days 1428 of the classical menstrual cycle Defined as the period between ovulation and the initiation of menstrual bleeding Characterized by high levels of both oestradiol and progesterone Corpus luteum The tissue formed after ovulation by thecal and granulosa cells from the remains of the collapsed ovarian follicle it is responsible for progesterone and oestradiol secretion during the secretory phase of the menstrual cycle In the absence of fertilization the corpus luteum degrades thus decreasing hormone synthesis and signalling the initiation of menstruation Pattern recognition receptors PRRs Multiple families of conserved receptors such as Tolllike receptors TLRs that are present on the cell surface or within intracellular compartments PRRs recognize conserved structures that are present on pathogens or that are produced as part of their life cycle Pathogen associated molecular patterns PAMPs Conserved structures that are an integral part of pathogens but not mammalian cells and that are recognized by patternrecognition receptors Examples include viral and bacterial components such as double and singlestranded RNA bacterial lipopolysaccharide and hypomethylated DNA Cervicovaginal lavage fluid CVL fluid The fluid recovered after gently washing the vaginal walls and external cervix it contains the cellular secretions present in the lower female reproductive tract Wira et al Page 14 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Tight junction proteins A 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of longitudinal studies AIDS 2006 207383 PubMed 16327322 144 Henning TR et al Increased susceptibility to vaginal simianhuman immunodeficiency virus transmission in pigtailed macaques coinfected with Chlamydia trachomatis and Trichomonas vaginalis J Infect Dis 2014 21012391247 PubMed 24755433 Wira et al Page 22 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 145 Schust DJ et al Potential mechanisms for increased HIV1 transmission across the endocervical epithelium during C trachomatis infection Curr HIV Res 2012 10218227 PubMed 22384841 146 Jarvis GA Chang TL Modulation of HIV transmission by Neisseria gonorrhoeae molecular and immunological aspects Curr HIV Res 2012 10211217 PubMed 22384840 147 Ghanem KG et al Influence of sex hormones HIV status and concomitant sexually transmitted infection on cervicovaginal inflammation J Infect Dis 2005 191358366 PubMed 15633094 148 Zervomanolakis I et al Physiology of upward transport in the human female genital tract Ann NY Acad Sci 2007 1101120 PubMed 17416925 Wira et al Page 23 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Box 1 Length and hormone dynamics of the menstrual cycle Only 10 of women have the classical 28day cycle consisting of 14day proliferative and secretory phases lengths of these phases in the remaining women range from 1023 days and 719 days respectively134 Given the marked changes in hormone levels that occur over short periods particularly at ovulation when oestradiol OE2 levels surge and recede in 7296 hours a shift of 23 days between menstrual cycles may represent a different endocrine environment in the female reproductive tract FRT There is also considerable variation in hormone levels between sequential menstrual cycles in an individual woman32135136 Multiple factors affect hormone responsiveness in the FRT including receptor expression coregulator expression and hormone concentration At the cellular level OE2 and progesterone P4 exert their effects via the intracellular and plasma membrane oestrogen and progesterone receptors which are expressed by multiple cell types within the FRT There are two cytoplasmic isoforms of each receptor the expression level of which in endometrial tissue peaks in the late proliferative phase before declining in the secretory phase137138 Hormone concentrations in the FRT are distinct from the peripheral circulation There is a steep gradient in hormone concentration between follicular fluid ovarian veins and peripheral circulation during the menstrual cycle with highest levels being detected in the ovaries and lowest levels in the general circulation For example in the proliferative phase OE2 levels reach more than 1500 ng ml1 in follicular fluid from the ovary containing the dominant follicle 1070 ng ml1 in its innervating ovarian vein 40250 ng ml1 in the opposite ovary and 00508 ng ml1 in its ovarian vein and in the peripheral circulation139140 Similarly in the secretory phase P4 concentration markedly increases in ovarian venous plasma 500 ng ml1 uterine circulation 20 ng ml1 and peripheral circulation 10 ng ml1 compared with in the proliferative phase 511 ng ml1141 Wira et al Page 24 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Box 2 STIs modify the window of vulnerability for HIV The presence of preexisting infections in the female reproductive tract can increase susceptibility to subsequent HIV infection142 and can potentially expand the window of vulnerability Mechanisms involved in this effect include increased inflammation upregulation of cytokine and chemokine expression and recruitment of susceptible target cells For example herpes simplex virus 2 infection increases the likelihood of acquiring HIV by 3 to 5fold143 Foci of CD4 T cells CD8 T cells and dendritic cells DCs expressing the HIV coreceptors CCchemokine receptor 5 CCR5 and DCspecific intercellular adhesion molecule 3grabbing nonintegrin DCSIGN form at the site of herpetic lesions and remain for several months after viral clearance and healing66 In ex vivo experiments these sites have a greater susceptibility to HIV infection Macaques coinfected with Chlamydia trachomatis and Trichonomas vaginalis become infected with simianhuman immunodeficiency virus SHIV during the proliferative phase of the menstrual cycle rather than during the secretory phase as has been described for animals without coinfections144 Women with chlamydial infection have an increased number of HIVsusceptible CD4CCR5 T cells in the endocervix compared with uninfected women145 Similarly gonococcal infection increases the number of CD4 T cells in the endocervix146 As CD4CCR5 T cells are the main target cells of HIV this could be one of the mechanisms by which chlamydial and gonococcal infections predispose women to HIV acquisition In addition analysis of cervicovaginal lavage fluid in women coinfected with HIV and C trachomatis showed an increased white blood cell count147 which suggests increased risk for HIV transmission These findings suggest that changes induced in the mucosal environment by sexually transmitted infections can overcome the immune defences that are normally present during the proliferative phase of the menstrual cycle Wira et al Page 25 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 1 Anatomy and histology of the FRT The female reproductive tract FRT is composed of distinct anatomical regions that undergo morphological changes during the menstrual cycle The lower FRT consists of the vagina and ectocervix and is protected by a stratified squamous epithelium which is composed of superficial intermediate and basal epithelial cells The thickness of the squamous epithelium remains fairly constant in humans during the menstrual cycle By contrast the upper FRT which consists of the endocervix endometrium and Fallopian tubes is covered by a singlelayer columnar epithelium In the endometrium the columnar Wira et al Page 26 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript epithelial cells proliferate during the menstrual cycle and form glands in the secretory phase The transformation zone is where the columnar epithelium of the upper FRT meets the squamous epithelium of the lower FRT Overlying the epithelial surface in the lower FRT and endocervix is mucus the consistency of which changes across the cycle becoming thick and viscous in the secretory phase Also present is a dynamic population of bacteria primarily composed of lactobacilli in most women that acidify the lumen of the lower FRT Underlying the epithelium is a dense layer of fibroblasts interspersed with immune cells T cells macrophages B cells neutrophils natural killer NK cells and dendritic cells DCs The transformation zone contains a particularly high number of immune cells compared with the rest of the FRT In the endometrium immune cells form lymphoid aggregates that reach peak size around ovulation and during the secretory phase of the cycle Wira et al Page 27 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 2 The menstrual cycle The 28day menstrual ovarian cycle is divided into four stages menstrual phase proliferative phase midcycle ovulation and secretory phase that are characterized by cyclic changes in hormone levels Day 0 is defined by the onset of menstrual bleeding which lasts for 35 days in most women Menses is followed by the proliferative phase during which the endometrial lining is reconstituted Folliclestimulating hormone FSH produced by the anterior pituitary gland induces oestradiol OE2 production by the ovary OE2 levels increase during the proliferative phase and peak before midcycle ovulation followed by a rapid drop in concentration Rising OE2 levels stimulate luteinizing hormone LH production by the anterior pituitary the levels of which surge in the lateproliferative phase within 2436 hours of the OE2 peak leading to ovulation and increasing progesterone P4 synthesis At the same time FSH levels increase by a smaller amount Both LH and FSH levels rapidly drop in the early secretory phase After ovulation the concentrations of P4 and to a lesser extent OE2 which are both produced by the corpus luteum in response to LH steadily increase before peaking at midsecretory phase Both FSH and LH levels remain low throughout the secretory phase In the absence of fertilization OE2 and P4 levels drop which leads to endometrial shedding and the onset of menses Immune changes in the FRT that occur as a result of cyclic changes in hormone levels create an optimal environment for successful fertilization and implantation during the secretory phase This environment of regulated immune responses creates a window of vulnerability during this phase with permissive conditions for the entry and survival of pathogens Wira et al Page 28 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 3 Fluctuations in immune cell populations in the FRT during the menstrual cycle Bars represent the mean standard error of the mean SEM from different studies analysing immune cell subsets in the female reproductive tract FRT endometrium5617273108 endocervix and ectocervix560616373 Studies included in the figure are limited to those that used flow cytometry as microscopic analysis of tissues does not enable immune cell frequency to be accurately obtained Statistical analyses are not possible because of the limited number of flow cytometry studies that have been carried out NK natural killer Wira et al Page 29 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 4 Oestradiolmediated control of interactions between epithelial cells fibroblasts and immune cells in the FRT Oestradiol OE2 functions directly through receptor expression on multiple cell types of the female reproductive tract FRT or indirectly through intermediary molecules to regulate gene transcription and protein expression and to alter the number distribution and phenotype of cells in the FRT OE2 induces the expression of multiple cytokines chemokines growth factors and antimicrobial proteins For example OE2mediated stimulation of epithelial cells increases the luminal secretion of antimicrobial proteins such as secretory leukocyte protease inhibitor SLPI elafin and human βdefensin 2 HBD2 in the uterus but decreases the secretion of elafin and HBD2 in the vagina possibly leading to differences in antiviral and antibacterial activity in the FRT lumen depending on anatomical location Transforming growth factorβ TGFβ granulocytemacrophage colony stimulating factor GMCSF macrophage colonystimulating factor MCSF and CC chemokine ligand 20 CCL20 are secreted by epithelial cells into the tissue environment under the influence of OE2 where they modulate immune cell chemotaxis and function for example leading to changes in dendritic cell DC responses to Tolllike receptor TLR ligands In contrast to its direct effects on epithelial cells OE2 can indirectly alter the proliferation and the barrier function of uterine epithelial cells by stimulating the secretion of hepatocyte growth factor HGF by uterine fibroblasts which in turn modulates tight junction expression and cell replication OE2 also directly affects uterine fibroblasts to increase their secretion of CCL2 and interleukin8 IL8 which leads to increased chemotaxis of neutrophils monocytes and DCs Less clear is the role of OE2 in regulating the contributions of immune cells to the mucosal environment in the FRT these cells secrete CCL5 tumour necrosis factor TNF and fibroblast growth factor 2 FGF2 TNF which is a proinflammatory cytokine activates fibroblasts and degrades tight junction integrity and Wira et al Page 30 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript thus the barrier function of epithelial cells Similarly FGF2 stimulates growth of uterine epithelial cells and fibroblasts and also alters epithelial structure and integrity not shown NK natural killer Wira et al Page 31 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 5 Influence of sex hormones on mucosal immunity in the lower and upper FRT during the window of vulnerability This figure depicts the key immunological mechanisms present in the female reproductive tract FRT that are essential for successful reproduction and that directly or indirectly affect pathogens that enter the FRT and threaten reproductive health These immune mechanisms are under hormonal control During the window of vulnerability oestradiol OE2 and progesterone P4 selectively stimulate andor suppress aspects of the innate and adaptive immune systems as shown in ways that vary according to the FRT site For example in the lower FRT innate components such as human βdefensin 2 HBD2 in the lumen are suppressed at a time when CD8 cytotoxic T lymphocyte CTL activity and natural killer NK cell cytotoxic activity are maintained By contrast CD8 CTL and NK cell activities are suppressed in the uterus at a time when luminal innate components are enhanced These uterine changes are consistent with increased luminal pathogen killing andor inactivation at a time when semiallogeneic blastocyst rejection might otherwise occur The resulting alterations in immune protection optimize conditions for successful implantation but also lead to an increased risk of acquiring sexually transmitted infections STIs3 In the table doubleheaded arrows indicate that there is no change CCL CCchemokine ligand CXCL12 CXCchemokine ligand 12 CX3CL1 CX3Cchemokine ligand 1 DC dendritic cell FcRn neonatal Fc receptor HGF hepatocyte growth factor IFNγ interferonγ IL8 interleukin8 pIgR polymeric IgA receptor SLPI secretory leukocyte protease inhibitor TGFβ transforming growth factorβ Wira et al Page 32 Nat Rev Immunol Author manuscript available in PMC 2016 January 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript