© 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

REVIEW The relationship between sex , the vaginal microbiome and immunity in HIV-1 susceptibility in women Jocelyn M. Wessels1,2, Allison M. Felker1,2, Haley A. Dupont1,2 and Charu Kaushic1,2,*

ABSTRACT approximately 1.5- to 5-times greater than via the male genital tract The role of sex hormones in regulating immune responses in the (1 in 2000 to 1 in 200 in females versus 1 in 3000 to 1 in 700 in female genital tract has been recognized for decades. More recently, males) (Hladik and McElrath, 2008). There are both socio- it has become increasingly clear that sex hormones regulate economic and biological reasons why women may be more susceptibility to sexually transmitted infections through direct and susceptible to STIs, including HIV-1, than men. The biological indirect mechanisms involving inflammation and immune responses. factors that could influence the outcome of pathogen exposure in the The reproductive cycle can influence simian/human immunodeficiency FGT include its large surface area, the alterations in physiology of virus (SHIV) infections in primates and HIV-1 infection in ex vivo reproductive tract tissues during different phases of the menstrual cervical tissues from women. Exogenous hormones, such as those cycle, the influence of sex hormones on mucosal immune defense, found in hormonal contraceptives, have come under intense scrutiny the use of hormonal contraceptives and the effect of the indigenous because of the increased susceptibility to sexually transmitted microbiota (see Box 1 for a glossary of terms). In this Review, we infections seen in women using medroxyprogesterone acetate, a highlight the mechanisms by which sex steroid hormones, including synthetic progestin-based contraceptive. Recent meta-analyses hormonal contraceptives, might impact the risk of HIV-1 concluded that medroxyprogesterone acetate enhanced HIV-1 susceptibility in women. This is an important and timely topic, susceptibility in women by 40%. In contrast, estradiol-containing given that approximately 40% of HIV-1 infections occur in the FGT hormonal contraceptives were not associated with increased and that women using the progestin-based injectable contraceptive susceptibility and some studies reported a protective effect of depot-medroxyprogesterone acetate (DMPA) are 40% more likely on HIV/SIV infection, although the underlying mechanisms to acquire HIV-1 than women not using hormonal contraceptives remain incompletely understood. Recent studies describe a key role for (Polis et al., 2016). The relevance of this area to public health is the vaginal microbiota in determining susceptibility to sexually emphasized by the fact that more than 8 million women in sub- transmitted infections, including HIV-1. While Lactobacillus spp.- Saharan Africa, where HIV-1 is endemic, use DMPA as their main dominated vaginal microbiota is associated with decreased form of contraception (Ross and Agwanda, 2012). susceptibility, complex microbiota, such as those seen in , correlates with increased susceptibility to HIV-1. The female genital tract Interestingly, sex hormones are inherently linked to microbiota The lower FGT, the vaginal tract and ectocervix (Box 1), is lined regulation in the vaginal tract. Estrogen has been postulated to play with epithelial cells covered by and colonized by bacteria. It a key role in establishing a Lactobacillus-dominated microenvironment, is the first location encountered by HIV-1 during heterosexual whereas medroxyprogesterone acetate is linked to hypo-estrogenic intercourse with an infected male partner. The lower FGT provides a effects. The aim of this Review is to contribute to a better understanding protective physical and immunological mucosal barrier. Acting as a of the sex-–microbiome–immunity axis, which can provide structural support beneath the is a dense layer of stromal key information on the determinants of HIV-1 susceptibility in the fibroblasts, in which a diverse population of leukocytes reside (Wira + female genital tract and, consequently, inform HIV-1 prevention et al., 2005). As HIV-1 preferentially infects CD4 leukocytes strategies. [T cells, macrophages and dendritic cells (DCs)] residing in the stroma, the vaginal mucus and epithelial barrier serve in part KEY WORDS: Vaginal microbiota, T cells, DMPA, Inflammation to impede viral access to target cells (Pope and Haase, 2003). In order for HIV-1 transmission to occur, infectious virions must Introduction transverse the protective physical and immunological barriers of the Clinical and experimental evidence indicates that many sexually FGT and infect target cells located in the stroma. While the exact transmitted infections (STIs) are more prevalent in women than men mechanisms by which HIV-1 accesses target cells remain (Kaushic et al., 2011). The probability of human immunodeficiency incompletely understood, this Review will focus on several virus (HIV) transmission via the female genital tract (FGT) is factors known to influence HIV-1 susceptibility and acquisition in the FGT. 1McMaster Immunology Research Centre, Department of Pathology and Molecular The lower FGT is lined with multi-layered squamous epithelial Medicine, Michael G. DeGroote Centre for Learning and Discovery, McMaster cells, and tight junctions linking these cells are mainly restricted to University, Hamilton, Ontario L8S 4L8, Canada. 2Department of Pathology and its basal layers. The epithelium in the lower FGT undergoes Molecular Medicine, McMaster University, Hamilton, Ontario L8S 4L8, Canada. continuous differentiation, resulting in a mitotically active basal *Author for correspondence ([email protected]) layer and a terminally differentiated superficial layer comprising cornified epithelial cells, which aid in preventing infection by C.K., 0000-0002-7088-2569 certain pathogens, including HIV-1 (Anderson et al., 2014). Aside This is an Open Access article distributed under the terms of the Creative Commons Attribution from acting as a physical barrier, the vaginal epithelial cells also License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. secrete mucins (Box 1) into the vaginal lumen. These form a Disease Models & Mechanisms

1 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Pattern recognition receptors (PRRs): part of the innate immune Box. 1. Glossary system, these protein sensors detect pathogen-associated molecular patterns (PAMPs) and induce an innate response in the host. 16S rRNA gene sequencing: an untargeted method to identify the bacterial taxa present in a microbiota. Typically, a variable region within RANTES (also known as CCL5): stands for ‘regulated on activation, the 16S ribosomal RNA (rRNA) gene that is unique to each taxa and normal T cell expressed and secreted’. A chemotactic protein codes for a component of the bacterial ribosome is amplified by PCR and (chemokine) that recruits T cells, eosinophils and basophils, or other sequenced. cells with cognate receptors.

Alpha-diversity: a term used in ecology to describe differences in Seroconversion: the point at which a specific antibody has developed species within a site, on a ‘local’ scale. In the context of this article, we are and become detectable in the peripheral blood (i.e. antibodies against referring to the unique diversity of bacterial species found in the vaginal HIV-1 indicate that seroconversion has occurred). microbiota of each woman in our study. Seronegative: a negative result in a blood test for antibodies against a Amsel criteria: a method to diagnose bacterial vaginosis (BV). certain virus or condition (i.e. HIV-1 or rheumatoid arthritis). Diagnosed by the presence of at least three of four clinical symptoms [thin/white/yellow vaginal discharge, clue cells (vaginal epithelial cells Toll-like receptors (TLRs): intra- or extracellular proteins that recognize that appear stippled owing to being covered with bacteria) on microbes and nucleic acids, and participate in the innate immune microscopy, vaginal pH>4.5, fishy odor following addition of potassium response. A type of pattern recognition receptor (PRR). hydroxide]. The Amsel criteria distinguish nonspecific (BV) from other forms of vaginitis and from normal findings, as defined in Amsel et al. (1983). hydrophobic layer of mucus that traps pathogens and prevents Antimicrobial peptides (AMPs): part of the innate immune response for access to the underlying epithelial cells (Lai et al., 2009; Quayle, host defense, found in all classes of life. 2002; Shukair et al., 2013). In addition to mucins, the vaginal mucus contains other host defense molecules, including antimicrobial Cervical transformation zone: the area of the where the peptides (AMPs; Box 1) and complement system components, columnar epithelial cells lining the endocervix change into the squamous epithelial cells lining the ectocervix; an area thought to be which can directly bind to and eliminate pathogens, impeding particularly prone to HIV-1 acquisition. access to the vaginal epithelium (Birse et al., 2015b; Quayle, 2002). These protective features can effectively dampen HIV-1 motility in Commensal: an organism living in relationship with another without human cervicovaginal mucus and enhance vaginal barrier function harming or benefitting the host organism. (Shukair et al., 2013). Colonizing the vaginal epithelium is an indigenous microbial community that can influence the physiology Defensins: small proteins found in vertebrates and invertebrates that function as antimicrobial host defense peptides. and immune function of the FGT (Cruickshank and Sharman, 1934; Ma et al., 2012). The resident vaginal microbiota (VMB) exists in a Dysbiosis: a microbial imbalance. mutualistic relationship with the female host and also participates in preventing vaginal infection by a variety of pathogens. Notably, Estradiol (E2)-based hormone replacement therapy (HRT): treatment disruption of the VMB is associated with an increased risk of STIs, with natural or synthetic alone or in combination with including HIV-1 (Atashili et al., 2008). The impact of the VMB on progestins, typically aimed at alleviating the symptoms of menopause, inflammation and susceptibility to HIV-1 is an emerging area of or preventing osteoporosis. interest and will be discussed in more detail below. Ectocervix: the outer portion of the cervix that is located within the The upper FGT consists of the endocervix, vaginal tract and lined by squamous epithelial cells. (), fallopian tubes and . It is lined by a single layer of epithelial cells linked by tight junctions that provide Elafin: a defensin (antimicrobial peptide) with antibacterial activity physical protection. It was originally believed that the upper FGT against bacterial and fungal pathogens. Also known as Trappin-2. was sterile. However, like the vaginal tract, the upper FGT is HIV-1 gp120: a glycoprotein found on the exposed surface of the HIV-1 colonized by bacteria (Chen et al., 2017; Eschenbach et al., 1986; envelope. Hemsell et al., 1989; Mitchell et al., 2015; Møller et al., 1995). Several studies have reported significant correlation in microbial Lactoferrin: a defensin (antimicrobial peptide) that also binds iron. community members across the FGT, but their relative proportions vary (Chen et al., 2017; Walther-António et al., 2016; Wee et al., Lysozyme: a defensin (antimicrobial protein) that enzymatically cleaves 2018). Furthermore, the quantity of bacteria found on the and thus damages the components of the cell wall of Gram-positive bacteria. endometrial surface are 2-4 log orders of magnitude lower than those found in the vaginal tract (Chen et al., 2017; Mitchell et al., Microbiota: a consortium of bacteria residing in and on multicellular 2015), and lactobacilli typically represent a large fraction of the organisms, including plants and animals. endometrial microbiota (Franasiak et al., 2016; Mitchell et al., 2015; Moreno et al., 2016; Wee et al., 2018). However, studies also Mucins: heavily glycosylated proteins (glycoproteins) produced identified many low-abundance genera (Moreno et al., 2016; by epithelial cells in most animals. They are a constituent of vaginal Verstraelen et al., 2016). Similar to the VMB, the endometrial mucus. microbiota may be able to modulate inflammation. In vitro co- NOD-like receptors: intracellular sensors of pathogen-associated cultures of endometrial epithelial cells with pathogenic bacteria molecular patterns (PAMPs). A type of pattern recognition receptor (Neisseria gonorrhoeae) induced proinflammatory mediators (PRR). (Christodoulides et al., 2000), whereas other bacteria typically found in the reproductive microbiotas (Lactobacillus crispatus, Disease Models & Mechanisms

2 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Gardnerella vaginalis) did not (Łaniewski et al., 2017). This expressed by microorganisms (Ghosh et al., 2013; Herbst- suggests that the endometrial microbiota may be able to modulate Kralovetz et al., 2008; Pioli et al., 2004). However, PRRs appear endometrial inflammation in the host. HIV-1 infection can occur in to be differentially regulated and expressed by epithelial cells the upper FGT, although the exact mechanism is unclear. HIV-1 has depending on their location within the FGT, with a limited been shown to induce innate inflammation that can disrupt the expression of PRRs in the lower compared to the upper FGT mucosal barrier in the upper FGT (Nazli et al., 2010), to transcytose (Ghosh et al., 2013; Hart et al., 2009; Pioli et al., 2004). Pathogen across the endometrial epithelial barrier (Ferreira et al., 2015; Hocini recognition by PRRs induces intracellular signaling and activates et al., 2001) and to infect ovarian tissue cells in vitro (Shen et al., the transcription factor NF-κB, resulting in the production of AMPs 2017), which could influence initiation of HIV-1 infection. Simian and pro-inflammatory cytokines (Fichorova et al., 2002; Nguyen immunodeficiency virus (SIV), the primate analog of HIV, can also et al., 2014). For instance, primary epithelial cells isolated from the rapidly access and infect target cells throughout the lower and upper human and ectocervix can induce NF-κB signaling upon FGT (Stieh et al., 2014). A number of other reviews have focused on activation of the TLR pathway to produce pro-inflammatory the possible mechanisms of HIV-1 infection in the upper FGT cytokines, including interleukin 6 (IL-6), IL-8, tumor necrosis (Dunbar et al., 2012; Ferreira et al., 2014; Hickey et al., 2011; Lee factor α (TNF-α), macrophage inflammatory protein 1α (MIP-1α) et al., 2015; Nguyen et al., 2014; Wira et al., 2015). In this Review, and MIP-1β (Fichorova et al., 2015, 2002). Indeed, epithelial cells we discuss the recent information that is relevant to HIV-1 from both the upper and lower FGT have been reported to activate transmission in the lower FGT (ectocervix and vagina), which signaling pathways following pathogen exposure (Ferreira et al., make up the major surface area exposed to HIV-1-infected semen 2013; Nazli et al., 2010, 2009). Furthermore, our group recently during heterosexual intercourse. demonstrated that HIV-1 gp120 (Box 1) signaling through TLR2 Although one of the main functions of the immune system in the induced interferon β (IFN-β) production in genital epithelial cells, FGT is to protect against an array of pathogens, it must concurrently which had an anti-inflammatory activity and protected the function allow for the main reproductive functions of the FGT, including of the epithelial barrier (Nazli et al., 2018). In addition to supporting sperm migration, oocyte fertilization and embryo inflammatory cytokines, cervicovaginal epithelial cells are known implantation. To balance these unique requirements, the FGT is to produce AMPs, including defensins (Box 1), secretory leukocyte precisely regulated by the sex steroid hormones estradiol (E2) and protease inhibitor (SLPI), lysozyme, lactoferrin and elafin (Box 1) , which are cyclically produced by the ovaries (Nguyen et al., 2014; Wira et al., 2005). In vitro cultures of throughout the menstrual cycle during the reproductive years in ectocervical epithelial cells secrete AMPs, including SLPI1, women (Reed and Carr, 2000; Wira et al., 2005, 2015). Both Trappin-2/elafin and human beta defensin 2 (HBD-2) (Wira et al., fluctuating endogenous sex hormones and hormonal contraceptives 2011), which are known to have anti-HIV-1 activity (Aboud et al., can alter the components of the FGT defensive barriers, including 2014; Ghosh et al., 2010; McNeely et al., 1995; Sun et al., 2005). mucus viscosity, epithelial barrier thickness, immune cell frequency Taken together, the epithelial cells of the lower FGT contribute to and resident vaginal microbes (Vitali et al., 2017; Wira et al., 2015). recognition of and defense against pathogens in part via their For example, the amount and composition of the vaginal mucus induction of inflammation and AMPs. varies with the menstrual cycle, hormonal contraceptive use, and Residing in the lower FGT are numerous, dynamic leukocyte hormone dampening at menopause (Chappell et al., 2015; Gipson populations that contribute to humoral and cellular immunity. et al., 1997; Shust et al., 2010). During ovulation, under the Leukocytes constitute 6-20% of all cells in the FGT (Nguyen et al., influence of estradiol, the vaginal mucus is thin with low viscosity, 2014; Wira et al., 2015), and possess unique characteristics that which facilitates sperm movement. Conversely, during the differentiate them from other tissue resident and peripheral leukocytes. progesterone-high luteal phase of the menstrual cycle, the vaginal For instance, flow cytometry measurements showed that primary mucus is thick and viscous, which impedes the movement of vaginal macrophages were more susceptible to HIV-1 infection particulates from the lower to the upper FGT. Together, the carefully in vitro than gastrointestinal macrophages (Shen et al., 2011). regulated fluctuations of these physicochemical features are Leukocytes of the FGT are preferentially distributed in necessary to promote both reproductive success and a robust immunological microenvironments depending on their function defensive barrier in the FGT. As these first lines of defense are within the FGT. In the cervical transformation zone (Box 1), T cells heavily influenced by both hormones and microbiota composition, and antigen-presenting cells (APCs), including many CD14+ their dynamic interactions can substantially influence HIV-1 macrophages and DCs, are particularly abundant (Pudney et al., susceptibility in women, and will be discussed in detail below. 2005; Trifonova et al., 2014). The presence of CD4+CCR5+ T cells, which are the primary targets for HIV-1 infection, renders the Immunity in the lower female genital tract transformation zone particularly vulnerable (Eslahpazir et al., 2008; In the lower FGT, epithelial cells, stromal fibroblasts and leukocytes Kumamoto and Iwasaki, 2012; Pudney et al., 2005; Trifonova et al., interact with each other, with the sex hormones and with the VMB 2014; Vitali et al., 2017). DCs, including intra-epithelial CD1a+ to induce the two of the immune response: innate and adaptive Langerhans cells, are present in the squamous epithelial cell layers of immunity (Ferreira et al., 2014; Nguyen et al., 2014; Zhou et al., the ectocervix and vagina (Pudney et al., 2005). These cells can be 2018). In addition to acting as a physical barrier against pathogens, infected by and are thought to facilitate HIV-1 transfer across epithelial the epithelial cells of the lower FGT modulate leukocyte function by cells of the lower FGT (Ballweber et al., 2011; Tchou et al., 2001). producing cytokines and chemokines, which induce leukocyte Further, cervical DCs express DC-SIGN, a C-type lectin receptor and differentiation and mediate inflammatory processes (Fahey et al., adhesion molecule that also acts as an HIV-1 receptor. The presence of 2005). Epithelial cells in both the upper and lower FGT (Fig. 1) DC-SIGN on DCs has been implicated in enhanced transmission of possess a wide array of pattern recognition receptors (PRRs), HIV-1 to T cells (Geijtenbeek et al., 2000; Geijtenbeek and van including Toll-like receptors (TLRs) and NOD-like receptors Kooyk, 2003). Thus, while T cells are the main targets for HIV-1, (Box 1), which recognize and respond to pathogens through macrophages and DCs can also be infected and therefore directly and conserved pathogen-associated molecular patterns (PAMPs) indirectly affect HIV-1 susceptibility in women. Disease Models & Mechanisms

3 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Lymphoid aggregate

Fallopian tube

Submucosa Tight Endometrium junctions Columnar epithelium Transformation zone

Cervix Langerhans cell

Vagina T cell Mucus

Antimicrobial peptides (AMPs)

Squamous ephithelium

Key

Dendritic cell Macrophage B cell CD8+ T cell CD56+ NK cell

CD4+ T cell TLR IgA IgG Lactobacilli

Fig. 1. Anatomy and immunological components of the female genital tract. The female genital tract (FGT) can be separated into the upper (, , uterus/endometrium and endocervix) and lower (ectocervix and vagina) tract. The vaginal epithelium has many innate immune protection mechanisms, such as tight junctions, antimicrobial peptides (AMPs) and mucus, in order to neutralize, trap and prevent entry of potential pathogens. The vaginal lumen is colonized by commensal bacteria, mainly lactobacilli, which help to maintain a low pH. Furthermore, immune cells such as γδ T cells, dendritic cells (DCs) and macrophages are present beneath and between the vaginal epithelial cell layer to survey the local environment for danger. The abrupt transition from keratinized squamous epithelial cells of the ectocervix to single columnar epithelial cells of the endocervix represents the transformation zone; this site has an abundance of HIV-1 target cells and has been proposed to be one of the major sites for infections. The presence of lymphoid aggregates in the endometrial tissue suggests that this is an inductive site for cell-mediated immunity. Lymphoid aggregates found beneath the endometrium are composed of B cells in the inner core surrounded by CD8+ CD4− T cells and an outer layer of macrophages. A scatter of CD56+ natural killer (NK) cells and CD4+ T cells could be found in between lymphoid aggregates. It was originally believed that the upper FGT was sterile; however, like the vaginal tract, the upper FGT is colonized by bacteria, including lactobacilli. Figure modified and reprinted with permission from Nguyen et al. (2014). TLR, Toll-like receptor.

Most T cells in the lower FGT reside near the basal epithelial cell the vagina and cervix possess cytolytic activity (Pudney et al., 2005; layer at the stromal cell interface; however, a large T-cell population White et al., 1997a). In recent years, T-helper 17 (Th17) cells have also resides in the ectocervical and vaginal epithelium as intra- been identified as preferential targets for HIV-1 in the human cervix epithelial lymphocytes (Pudney et al., 2005). Although CD8+ T (Cicala et al., 2009; McKinnon et al., 2011, 2015). These cells are slightly more abundant than CD4+ T cells (Trifonova et al., CD4+IL17A+ cells co-express the HIV-1 co-receptor CCR5 and 2014), the CD4+ T cells represent a greater number of the intra- mucosal integrin α4β7, which is known to bind HIV-1 gp120 and epithelial lymphocytes in the ectocervix (Pudney et al., 2005). enhance viral dissemination (Cicala et al., 2009). Importantly, this Interestingly, the number of vaginal intra-epithelial CD4+ and CD8+ Th17 population is almost completely depleted in HIV-1+ women, T cells, as well as CD1a+ DCs, is elevated in women with indicating its potential as a key target for HIV-1 transmission + inflammation of the FGT (Pudney et al., 2005), and CD8 T cells of (McKinnon et al., 2011). Thus, given their abundance, Disease Models & Mechanisms

4 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147 susceptibility and location, CD4+ T and Th17 cells are considered Effect of endogenous and exogenous sex steroid hormones the major targets of HIV-1 in the lower FGT. Hence, the factors on immunity in the FGT influencing the number and activation of these cells can greatly It is well documented that the endogenous female sex steroid impact HIV-1 susceptibility. hormones estradiol and progesterone, and their synthetic analogs, Although B cells are a key cellular component of the adaptive including those found in hormonal contraceptives, regulate immunity immune system, they are not present in large numbers in the lower in the FGT. Other reviews provide a detailed account of this FGT. Resident immunoglobulin A (IgA)-expressing plasma cells immunoregulatory influence on immune cell phenotype and function have been described in the cervix, especially in the endocervix (Dunbar et al., 2012; Kaushic, 2011; Nguyen et al., 2014; Vitali et al., (Nguyen et al., 2014). In fact, studies examining antibody responses 2017; Wira et al., 2014). In this article, we focus on the effect of in the FGT have identified modest IgA and IgG responses against endogenous and exogenous hormones in modulating key target cell viral infections, including HIV-1, in cervical secretions (Russell and populations, and thus HIV-1 susceptibility, in the lower FGT. Mestecky, 2002). Unlike in other mucosal sites, the dominant Although alterations in immune cell populations occur in antibody isoform produced during the adaptive immune response in endometrial tissues of the upper FGT (Nguyen et al., 2014), flow the FGT is IgG rather than IgA (Li et al., 2011; Russell and cytometry analyses did not confirm major fluctuations in their Mestecky, 2002), yet both isoforms can transfer across the genital location or abundance in the lower FGT between the phases of the epithelium. In the case of IgG, the neonatal Fc receptor (FcRN) on menstrual cycle or between pre- and post-menopausal women genital epithelial cells facilitates transfer of IgG to the lumen to (Givan et al., 1997; Pudney et al., 2005; Trifonova et al., 2014). confer protection against vaginal infections (Li et al., 2011). Even High levels of cytolytic activity were observed in CD3+ T cells though relatively little is known about the role of B cells and isolated from the cervix and vagina, but this was independent of antibodies in the FGT, in theory they have the potential to contribute menstrual cycle stage. Women of reproductive age have to adaptive immunity and offer an additional layer of protection significantly more CD4+, CD8+ and B cells in their ectocervix against HIV-1. An overall summary of the general changes in than in the endocervix (Trifonova et al., 2014). Further, the immunity over the menstrual cycle are highlighted in Fig. 2. ectocervix has also been shown to contain higher numbers of CD4+

Menstrual cycle DMPA

Follicular phase Luteal phase

Progestin

Estradiol

Progesterone Estradiol

Days 0 7 14 21 28 0 20 90

Estradiol Progesterone Progestin (MPA)

Expression of CXCR4, CCR5 Inflammatory cytokine production Th2 cytokine response Inflammatory cytokine production Th17 differentiation CD1a+ Langerhans cells CCR5+CXCR4+CD4+ target cells DC-mediated antigen uptake DC-mediated antigen uptake Induction of T-cell proliferation by DCs Antimicrobial peptides Antimicrobial peptides Antimicrobial peptides IgG, IgA secretion IgG, IgA secretion

HIV susceptibility HIV susceptibility HIV susceptibility

Fig. 2. Changes in lower female genital tract immunity and HIV-1 susceptibility under endogenous and exogenous sex hormones. The endogenous levels of female sex hormones (estradiol and progesterone) vary throughout the 28-day menstrual cycle in women. Estradiol dominates the follicular phase and reaches peak levels just prior to ovulation, which occurs around day 14. Post-ovulation, estradiol levels decline as progesterone levels rise towards the mid- luteal phase. The progestin-based injectable contraceptive depot-medroxyprogesterone acetate (DMPA) is administered every 3 months. Levels of this progestin, a synthetic progesterone, are highest in serum within the first 20 days of administration. Women on DMPA have consistently lower estradiol levels than normally cycling women (Hapgood et al., 2018). A summary of the putative effects of hormones and hormonal contraceptives on female genital tract (FGT) immunity as presented in this Review are summarized as shown. Overall, while estradiol appears to promote factors related to decreased HIV-1 susceptibility, alterations in immunity during periods of high progesterone or DMPA use are associated with increased HIV-1 susceptibility. DCs, dendritic cells; Ig, immunoglobulin; Th2, T-helper type 2 cells; Th17, T-helper 17 cells. Disease Models & Mechanisms

5 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

T cells in women with vaginal/cervical inflammation (Pudney et al., In addition to the impact of endogenous sex steroid hormones on 2005; White et al., 1997b). However, other phenotypic and functional immunity in the FGT, exogenous hormones, such as those changes can occur within specific immune cell populations in commonly found in hormonal contraceptives, can also affect response to sex hormones. For example, cellular proliferation and immunity in the FGT. This is an area of active research, particularly secretion of Th1-type cytokines was impaired in peripheral blood because the progestin-based injectable contraceptive DMPA is CD4+ and CD8+ T cells treated with progesterone, instead favoring the linked to increased HIV-1 susceptibility (Hapgood et al., 2018; development of Th2-type cytokine-producing T cells (Enomoto et al., Kaushic, 2009; Morrison et al., 2015; Polis et al., 2016). In a recent 2007; Piccinni et al., 1995). Progesterone treatment of murine bone- study, women on DMPA had greater expression of RANTES (also marrow-derived DCs resulted in inhibited differentiation of DCs, known as CCL5; Box 1) and decreased expression of the AMP leading to increased antigen uptake and decreased production of BD2, which were implicated in HIV-1 seroconversion (Box 1) inflammatory cytokines, whereas opposing effects were observed (Fichorova et al., 2015). Furthermore, an increased frequency of the after estradiol treatment (Xiu et al., 2016). Furthermore, HIV-1 target CCR5+CD4+ T cells was also observed in cervical cell immunohistochemistry of vaginal biopsies from women of populations isolated from women using injectable progestin-only reproductive age who received intravaginal delivery of exogenous contraceptives, including DMPA (Byrne et al., 2016; Chandra et al., progesterone showed increased numbers of CD1a+ Langerhans cells 2013). In contrast, women using a progestin-based intrauterine (Wieser et al., 2001), which are implicated in HIV-1 transfer across device (IUD) had reduced expression of the HIV-1 co-receptor epithelial cell barriers (Ballweber et al., 2011; Tchou et al., 2001). In CCR5 on cervical T cells (Achilles et al., 2014). However, increased addition to affecting HIV-1 target (CD4+HLA-DR+CD38+CCR5+) numbers of HIV-1 target cells are not consistently observed in cells, progesterone also alters humoral immunity, as IgG and IgA women on DMPA (Michel et al., 2015; Mitchell et al., 2014). MPA, titers in the cervical secretions were diminished in the luteal compared the active component in DMPA, was also shown to inhibit the to the follicular phase of the menstrual cycle (Safaeian et al., 2009), activation of T cells and peripheral dendritic cells (pDCs) in and immunoglobulin levels peaked just prior to ovulation, when response to T-cell-receptor- and TLR-mediated activation at estradiol levels were high (Franklin and Kutteh, 1999; Kutteh et al., physiological concentrations in peripheral blood mononuclear 1998). Similarly, progesterone is elevated in the luteal phase and is cells derived from pre-menopausal women (Huijbregts et al., correlated with increased secretion of AMPs including defensins, 2014). In other studies, MPA also impaired the expression of DC trappin-2/elafin and SLPI in cervicovaginal lavage (Dunbar et al., activation markers and affected their ability to promote T-cell 2012; Ghosh et al., 2010). Collectively, these results help illuminate proliferation (Quispe Calla et al., 2015). Recent transcriptomic the possible mechanisms by which progesterone might modulate studies from our group investigated the impact of sex hormones and immunity in the FGT and relate to HIV-1 susceptibility, which is MPA on the mRNA profile of primary endometrial epithelial cells greater in women in the progesterone-high luteal phase of the and found that MPA significantly increased expression of menstrual cycle (Saba et al., 2013; Wira and Fahey, 2008). inflammation-related genes (Woods et al., 2018). Taken together, Although progesterone has been linked to increased HIV-1 MPA is thought to negatively impact immunity as it relates to HIV-1 susceptibility, estradiol can also impact immunity in the FGT. susceptibility in the FGT, despite several studies reporting it to be Depending on its concentration, estradiol can exert pro- or anti- immunosuppressive (Hapgood et al., 2018). The overall changes in inflammatory responses and differentially affect immune function immunity under the influence of DMPA are highlighted in Fig. 2. (Straub, 2007). Both sex steroid hormones can also affect PRR Combined oral contraceptives (COCs), which contain analogs expression throughout the FGT. Estradiol is able to modulate the of both estradiol and progesterone, are generally thought to have downstream signaling of PRRs and pro-inflammatory receptors, as protective effects against viral infection in the FGT, depending on well as regulate NF-κB function (Fahey et al., 2008; Ghisletti et al., their formulations and quantity of the analogs. For example, one 2005; Schaefer et al., 2005). For instance, estradiol inhibits the study found higher levels of inflammatory cytokines, including mRNA expression of inflammatory cytokines IL-1α, Il-6, IL-8 and IL-1β, IL-6 and IL-8, in women on COCs compared to women TNF-α in vaginal epithelial cell lines (Wagner and Johnson, 2012). who were not using hormonal contraceptives (Fichorova et al., Similarly, in vitro studies of human ectocervical epithelial cells 2015). While T cells isolated from the cervical epithelium of treated with estradiol showed decreased production of IL-1β and womenusingCOCsshowednodifferenceinexpressionof IFN-γ (S Lashkari and Anumba, 2017), and vaginal cells captured activation markers or CXCR4 compared to women not on COCs, using a menstrual cup and treated with estradiol had decreased the number of T cells expressing CCR5 was higher in women on secretion of the AMPs HBD2 and elafin (Patel et al., 2013). In COCs (Prakash et al., 2002). Conversely, multiple studies primary murine microglia and RAW 264.7 cell macrophage investigating immunoglobulins in cervical secretions revealed populations, treatment with estradiol induced anti-inflammatory that women on COCs had higher IgG and IgA levels than naturally activity by inhibiting intracellular transport of NF-κB family cycling women (Franklin and Kutteh, 1999; Safaeian et al., 2009), members to the nucleus (Ghisletti et al., 2005), while exogenous suggesting that adaptive immunity might be stronger in the women estradiol treatment of peripheral blood macrophages and CD4+ T on COCs. Thus, exogenous exposure to hormonal contraceptives cells reduced HIV-1 viral entry through a CCR5-independent impacts immunity in the lower FGT, in ways that may impact pathway (Rodriguez-Garcia et al., 2013). Estradiol can also inhibit susceptibility to HIV-1 infection. in vitro Th17 differentiation in mouse splenocytes (Chen et al., 2015), and mouse models of viral infection have demonstrated that Factors affecting susceptibility to HIV-1 in women estradiol can enhance the response of Th17 cells and Th1-type To establish infection in the FGT, HIV-1 must first evade the responses following viral exposure (Anipindi et al., 2016). Taken previously discussed anatomical and biological barriers. Although together, these results suggest that estradiol typically maintains an these are effective, since the frequency of HIV-1 infection is anti-inflammatory immune environment that helps reduce approximately 1 in 200 to 1 in 2000 per act of unprotected receptive susceptibility to infections while still maintaining the ability to vaginal intercourse (Hladik and McElrath, 2008), several factors can mount an inflammatory, anti-viral response when necessary. influence susceptibility (Ferreira et al., 2014). Firstly, disruption of Disease Models & Mechanisms

6 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147 the vaginal epithelial barrier can enhance HIV-1 susceptibility in barrier by maintaining ZO-1 and occludin when primary upper women. HIV-1 can penetrate the squamous epithelium of the lower FGT cells were exposed to HIV-1 and gp120 (Ferreira et al., 2015). FGT through simple diffusive percolation, and the depth of viral Although an inflammatory milieu is believed to be a primary penetration significantly increases when the epithelial tight contributor to HIV-1 susceptibility in women, the frequency of junctions are disrupted (Carias et al., 2013). Disruption of the vaginal HIV-1 target cells was found to be the key predictor of vaginal epithelial barrier can occur during heterosexual intercourse HIV-1 susceptibility in a humanized mouse model of heterosexual as a result of microabrasions. These activate local inflammation and transmission, independent of inflammatory cytokines (Nguyen wound-healing processes, and promote infiltration of immune cells, et al., 2017). These results suggest that increased numbers of HIV-1 which could ultimately amplify the population of HIV-1 target cells target cells are sufficient to increase susceptibility, even without (Miller and Shattock, 2003; Norvell et al., 1984). In fact, women enhanced pro-inflammatory cytokine production. However, the with disruptions of the genital epithelium are at the greatest risk of presence of inflammation significantly increases HIV-1 HIV-1 acquisition (hazard ratio of 4.30) compared to women susceptibility. without compromised epithelial barriers (Abbai et al., 2016), further As mentioned in the previous section, endogenous sex steroid supporting the association between barrier disruption and HIV-1 hormones can modulate immunity in the FGT. Thus, in addition to acquisition. Alternatively, the epithelial barrier can be disrupted by the integrity of the vaginal barrier and inflammation, sex steroid HIV-1 and its gp120, which disrupt tight-junction proteins like ZO- hormones and, by extension, hormonal contraceptives, represent 1 and occludin in cultured primary upper FGT cells (Nazli et al., another factor that can modify susceptibility to HIV-1 in women. 2010, 2013). In vivo studies have also shown that differing Generally, estradiol has been associated with protection against hormonal and microbial compositions influence protease levels in HIV-1, while progesterone and the synthetic progestin DMPA the FGT, which may in turn impact the integrity of the vaginal appear to increase risk (Birse et al., 2015a; Morrison et al., 2015; epithelial barrier and influence HIV-1 susceptibility (Arnold et al., Saba et al., 2013). In fact, some studies have found significantly 2016; Birse et al., 2015a; Borgdorff et al., 2016). Normally, the low increased target T cells in women on DMPA (Byrne et al., 2016; pH of the vagina provides another layer of protection against Chandra et al., 2013). However, because this observation is not pathogens. However, the alkaline pH of semen temporarily consistent (Michel et al., 2015; Mitchell et al., 2014), it cannot be increases vaginal pH, and this can prevent HIV-1 from being the mechanism by which DMPA enhances susceptibility to neutralized by the normally acidic cervicovaginal mucus (Lai et al., HIV-1. Not only are sex hormones and hormonal contraceptives 2009; Tevi-Bénissan et al., 1997), thereby increasing the risk of thought to manipulate local HIV-1 target cells, but recent studies infection. Therefore, mechanisms that breach the vaginal epithelial have also implicated them in epithelial tissue remodeling, immune barrier or alter the pH can significantly increase the odds of HIV-1 cell migration and microbiota composition, suggesting they can successfully encountering its target cells. impact more than one of the factors that contribute to HIV-1 There is also a strong epidemiological association between susceptibility (Achilles et al., 2018; Mitchell et al., 2014; Woods concurrent STIs and increased risk of HIV-1 acquisition; in meta- et al., 2018). analyses, both human papillomavirus (HPV) and herpes simplex A more recently established risk factor for HIV-1 that we virus 2 (HSV-2) have been associated with a 2- to 3-fold increased highlight in this Review is the VMB. Disruption of the resident risk of HIV-1 acquisition (Freeman et al., 2006; Houlihan et al., VMB can be linked to increased HIV-1 susceptibility, and is most 2012). While the precise mechanisms for these epidemiological notable in the case of bacterial vaginosis (BV). In fact, BV has associations remain unclear, research suggests that the augmented been shown to increase the frequency of HIV-1 infection up to risk is due in part to increased inflammation in the FGT, as shown by 60% (Atashili et al., 2008). Although increased relative abundance higher inflammatory cytokines present in primary genital epithelial of anaerobes and decreased numbers of lactobacilli are key features cells exposed to co-pathogens (Ferreira et al., 2011), which of BV, the mechanisms by which the VMB increases HIV-1 enhanced HIV-1 infection and replication in target cells. In susceptibility remain largely unknown, but are speculated to relate support of this hypothesis, genital HSV-2 infection has been to their control of vaginal inflammation. Although the etiology and found to induce a pro-inflammatory profile characterized by bacterial species that initiate BV are widely debated, it has been increased numbers of HIV-1 target cells that can persist at proposed that BV might be a biofilm condition (Muzny and mucosal sites of HSV-2 reactivation, which may explain in part Schwebke, 2016; Schwebke et al., 2014). It has been hypothesized why individuals with HSV-2 have increased HIV-1 susceptibility that G. vaginalis, which can be present in the VMB of women (Zhu et al., 2009). In general, any state resulting in the activation of without BV, can induce formation of a biofilm that includes other the immune system and/or the recruitment of immune cells, BV-associated bacteria (Schwebke et al., 2014). The BV biofilm including HIV-1 target cells, may enhance the risk of HIV-1 adheres to the vaginal epithelium and produces cytotoxic susceptibility. In fact, a recent study found that the risk of HIV-1 substances (Muzny and Schwebke, 2016), and may also impact acquisition was increased 3-fold in women with genital local immune function and thus HIV-1 susceptibility. BV often inflammation, as defined by elevated HIV-1 target-cell-recruiting recurs or is refractive to antibiotic treatment, perhaps due to chemokines and inflammatory cytokines (Masson et al., 2015). persistence of the biofilm. It is however important to note that Conversely, reduced inflammation in the FGT was associated with biofilms can be present in women without BV [as assessed by the protection from HIV-1. In a cohort of women who were highly Amsel criteria (Amsel et al., 1983); Box 1] (Reid, 2018; exposed to HIV-1 yet remained seronegative (Box 1), dampened Swidsinski et al., 2014), and the effect of these biofilms on genital immunity was proposed to limit target cell availability and vaginal immunity is largely unknown. While many factors activation, and to maintain the protective vaginal barrier, thus associated with increasing the risk of HIV-1 in women have decreasing the risk of HIV-1 infecting its target cell population been described, including disruption of the vaginal barrier, (Lajoie et al., 2012). This relationship has also been demonstrated in concurrent STIs, sex hormones/hormonal contraceptives and the vitro, where pre-treatment with curcumin, a potent anti- VMB, HIV-1 infection is multifactorial and likely involves inflammatory agent, prevented the disruption of the mucosal mechanisms that remain to be elucidated (Fig. 3). Disease Models & Mechanisms

7 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Bacterial vaginosis As mentioned previously, certain species of lactobacilli appear to be Wound protective against HIV-1, and prospective studies have shown that Barrier permeability healing women with Lactobacillus-dominant VMB are not as likely to acquire Sex hormones HIV-1 as those with more diverse VMB (Gosmann et al., 2017; Low et al., 2011; Nunn et al., 2015). However, the species of Lactobacillus Other STIs seems important, as Lactobacillus iners did not have as strong of a Increased co-receptors protective effect against HIV-1 as other species of lactobacilli DMPA Inflammation (Gosmann et al., 2017). The main mechanism by which vaginal Target cells bacteriaarebelievedtomodifyHIV-1riskisbyalteringlocal inflammation and HIV-1 target cell populations (CD4+HLA- Hormonal contraceptives DR+CD38+CCR5+ cells) (Gosmann et al., 2017; Lennard et al., Vaginal microbiota 2017). At present, it is difficult to discern the precise mechanism by Vaginal pH which vaginal bacteria manipulate HIV-1 target cells within the vaginal mucosa. However, in vitro bacterial co-culture with VK2/E6E7 vaginal Cell activation epithelial cells has shown that certain bacterial genera (Fusobacterium, Fig. 3. Factors affecting susceptibility to HIV-1 in women. There are many Aerococcus, Sneathia, Gemella, Mobiluncus and Prevotella) induce factors associated with an increased risk of HIV-1 in women, including secretion of pro-inflammatory cytokines, including IL-1α,IL-1β,TNF- disruption of the vaginal barrier, concurrent STIs, the sex hormones, hormonal α, IL-8 and RANTES, by activating cellular TLRs (Anahtar et al., contraceptives and the vaginal microbiota (VMB). However, the establishment 2015; Fichorova et al., 2011). Conversely, pro-inflammatory cytokines of HIV-1 infection is multifactorial and likely involves mechanisms that remain are not induced when vaginal epithelial cells are co-cultured with L. to be elucidated. A word cloud was created using wordclouds.com to depict factors associated with increasing susceptibility to HIV-1 in women. The size of crispatus (considered to be protective against HIV-1) or other vaginal the text is proportional to the number of publications returned in PubMed commensals (Box 1) (Doerflinger et al., 2014; Rose et al., 2012), (March 2018) when searching for HIV-1 and the corresponding factor. Factors suggesting that epithelial cells sense bacteria via TLRs in a species- returning 25-99 hits (i.e. hormonal contraceptives) are listed in the smallest specific manner. Clinical studies have demonstrated direct correlations font, followed by factors returning 100-499 hits (i.e. sex hormones), 500-999 between non-Lactobacillus-dominant VMBs and increased levels of hits (i.e. target cells), and the largest font represents factors with more than inflammatory cyto- and chemokines in the vagina, including TNF-α, 1000 hits (i.e. inflammation). IFN-γ,IL-1α,IL-1β, IL-8, IL-10, IL-17, IL-23, MIP-1α and MIP-1β (Anahtar et al., 2015; Gosmann et al., 2017), supporting the in vitro results. Genital APCs also sense vaginal microbes. Phenotypic and Effects of the vaginal microbiota on inflammation, immunity transcriptional profiling of genital APCs collected from women with and susceptibility to HIV-1 highly diverse VMBs demonstrated that APCs likely contribute to The indigenous microbiota lining mucosal tissues in the human genital inflammation by responding via TLR-4, activating NF-κB, body modulate physiology and immunity at these sites. Although inducing chemokine secretion and recruiting HIV-1 target cells the majority of research demonstrating microbial manipulation of (Anahtar et al., 2015). Thus, vaginal bacteria sensed by genital host immune responses has been conducted in the intestinal tract epithelial cells and APCs likely manipulate vaginal inflammation, and and is reviewed elsewhere (Arranz et al., 2013; Jacobs and Braun, can thereby influence susceptibility to HIV-1. 2014; Round and Mazmanian, 2009), increasing evidence suggests In addition to enhancing HIV-1 target cell recruitment, another that the VMB plays similar immunomodulatory roles. Unlike the mechanism by which vaginal bacteria might enhance susceptibility gut microbiome, in which bacterial diversity is associated with to HIV-1 is via the breakdown of the vaginal epithelial barrier. health (Kinross et al., 2011), the VMB tends to have low diversity As previously mentioned, vaginal bacteria can induce pro- and be predominantly composed of Lactobacillus species (Ravel inflammatory cytokines. When primary genital epithelial cells et al., 2011). Lactobacillus crispatus in particular appears to respond to in vitro HIV-1 infection, they secrete pro-inflammatory correlate with protection against STIs and adverse reproductive cytokines (TNF-α, IL-6, IL-8, IP-10, RANTES), which results in outcomes via a variety of mechanisms reviewed elsewhere (Anahtar decreased trans-epithelial resistance (which is a measure of et al., 2018; Mirmonsef and Spear, 2014; Petrova et al., 2015). epithelial barrier integrity), disruption of ZO-1 and occludin, and However, the composition of the VMB varies by ethnicity (Zhou increased leakage of blue dextran dye across the cellular monolayer. et al., 2007); 80-90% of Caucasian and Asian women and 60% of This suggests that vaginal bacteria may also impair mucosal barrier black and Hispanic women typically have Lactobacillus-dominant function via their induction of pro-inflammatory cytokines (Ferreira VMBs (Ravel et al., 2011). This suggests that host genetics might be et al., 2015; Nazli et al., 2010). Additional evidence supporting the capable, at least in part, of affecting the bacterial species that involvement of the VMB in vaginal epithelial barrier impairment colonize the FGT, perhaps via subtle differences in vaginal can be gleaned from in vivo studies linking dysbiosis (Box 1) and immunity. It is however important to note that the relationship increased inflammatory cytokines with altered vaginal proteases between the VMB and ethnicity is complex and might rather reflect and mucosal proteins (Arnold et al., 2016; Borgdorff et al., 2016). differences in vaginal hygiene practices, a differential risk of other This link was stronger during the luteal phase of the menstrual STIs and/or sexual networks, amongst other factors (Birse et al., cycle, when progesterone is high (Birse et al., 2015a). Furthermore, 2017; Bradshaw et al., 2014; Brotman et al., 2008; Chaban impairing the integrity of the vaginal epithelial barrier allows for et al., 2014; Eschenbach et al., 2000; Forcey et al., 2015; Gajer et al., microbial translocation (Nazli et al., 2010), and this might further 2012; Marrazzo et al., 2002; Neggers et al., 2007; Schwebke et al., perpetuate the recruitment and activation of immune cells, including 1999; Vodstrcil et al., 2017; Wessels et al., 2017). Taken together, HIV-1 target cells, above the level that would otherwise occur as a ethnicity alone is not likely to explain the composition of the VMB result of bacterial sensing by epithelial and APC TLRs alone. Taken in individual women, and a comprehensive review of the factors together, the VMB can manipulate susceptibility to HIV-1 by affecting VMB composition is warranted. modifying both inflammation and integrity of the FGT barrier. Disease Models & Mechanisms

8 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Effect of hormones on the vaginal microbiota not use hormonal contraceptives (Morrison et al., 2015; Polis et al., Researchers can glean undeniable evidence for the role of sex 2016). In 2015, 56 million women worldwide used injectable hormones in shaping the composition of the VMB from studying hormonal contraceptives (United Nations Department of Economic pubertal girls, women at menopause, animal models and in vitro and Social Affairs Population Division, 2015). Therefore, bacterial co-culture. Although the VMB is relatively stable, the major understanding their impact on vaginal microbes is of great hormonal shifts that occur around (Gerstner et al., 1982; importance. Indeed, a recent study found that the strongest Hickey et al., 2015; Hill et al., 1995) significantly change its independent predictors of genital inflammation were use of composition from mainly anaerobic bacteria (Alvarez-Olmos et al., hormonal contraceptives (all methods grouped) and a VMB 2004) to one dominated by lactobacilli. At menopause, when subtype that predominantly included women with high Nugent estrogen levels decrease significantly, the VMB is less likely to be scores (indicating high microbial diversity) (Lennard et al., 2017). dominated by lactobacilli than in pre- and peri-menopausal women. These findings suggest that both hormonal contraceptives and the Post-menopausal women were at 7.8-times greater odds of being vaginal microbes are able to modify HIV-1 target cells in the vaginal colonized by a diverse array of bacteria than pre-menopausal women mucosa. Previous studies that only examined a targeted subset of the (Brotman et al., 2014b; Shen et al., 2016). Estradiol is thought to be a VMB and did not employ 16S rRNA gene sequencing (Box 1) did main driver in shifting the VMB towards Lactobacillus dominance, not find use of hormonal contraceptives to be a confounder of VMB although the mechanisms by which this occurs remain incompletely composition (Borgdorff et al., 2015), and showed that women on understood. Estradiol-based hormone replacement therapy (HRT; hormonal contraceptives were more likely to have Lactobacillus Box 1) maintains Lactobacillus dominance in post-menopausal fermentum in their VMB than those who were not (Kazi et al., women (Brotman et al., 2014b; Ginkel et al., 1993; Shen et al., 2016), 2012). Moreover, the initiation of DMPA injections decreased G. supporting a link between estradiol and lactobacilli. Additionally, vaginalis and total bacterial load (Roxby et al., 2016), and it also estradiol can increase adhesion of lactobacilli to epithelial cells (Silva reduced the proportion of women harboring H2O2-producing et al., 2004), and was proposed to enhance glycogen deposition in the Lactobacillus (Mitchell et al., 2014). Additionally, a quantitative- human vaginal epithelium (Cruickshank and Sharman, 1934; Farage PCR-based study quantified five Lactobacillus and four BV- and Maibach, 2006), via an unknown mechanism. Glycogen, a associated species to demonstrate that DMPA was associated with glucose polysaccharide and an important nutrient for lactobacilli lower quantities of lactobacilli compared to women not using (Cruickshank and Sharman, 1934; Mirmonsef et al., 2014; Paavonen, hormonal contraceptives, even excluding BV as a potential 1983), can be synthesized by vaginal epithelial cells and released into confounder (Jespers et al., 2017). Although the aforementioned the vaginal mucus (Fig. 1). Experimental administration of estradiol studies included women on a variety of systemically administered can induce glycogen deposition in vaginal tissues in hamsters and hormonal contraceptives such as injectables, oral pills, patches, etc., non-human primates (Gregoire and Parakkal, 1972; Gregoire and emerging data suggests that estrogen-containing vaginal rings, Richardson, 1970) via an unknown mechanism. The availability of which are topically administered, may have a more profound effect glycogen in the epithelial cells is thought to select for vaginal on vaginal lactobacilli due to their proximity in the vaginal tract (De colonization by microbes capable of metabolizing it. However, the Seta et al., 2012; Hardy et al., 2017; Huang et al., 2015; Veres et al., relationship between estradiol, free glycogen released into the vaginal 2004). Thus, the of hormonal contraceptives lumen by the epithelial cells and lactobacilli may not be as simple, as is likely to differentially impact the VMB, and this should be kept in recent studies did not find a direct correlation between circulating mind for multi-purpose HIV-1 prevention strategies aimed at peripheral estradiol and free glycogen (Mirmonsef et al., 2016; combining antiretroviral therapy and hormonal contraceptives. Mirmonsef and Spear, 2014), and few, if any, species of vaginal Next-generation 16S rRNA gene sequencing of the VMB lactobacilli directly metabolize glycogen (Martín et al., 2008; Nunn demonstrated that VMBs from individual women do not cluster and Forney, 2016; Spear et al., 2014; Stewart-Tull, 1964). The lack of together by method of contraception (Birse et al., 2017; Byrne et al., correlation between estradiol and free glycogen could be due to the 2016), suggesting that, although hormonal contraceptives did not fact that estradiol concentrations fluctuate rapidly over the menstrual cause major changes in the VMB, subtle changes may be cycle, peripheral estradiol concentrations are generally lower than biologically relevant. Indeed, a recent 16S rRNA sequencing those in the FGT, and free glycogen might be differentially utilized or study found that women on oral contraceptives had enhanced L. degraded depending on the composition of the VMB (Mirmonsef crispatus and decreased BV-associated bacteria loads (Brooks et al., et al., 2016). Furthermore, α-amylase, an enzyme that cleaves 2017), supporting the findings of meta-analyses showing that glycogen into simple sugars, has been isolated in fluid from the lower hormonal contraceptives can reduce the risk of BV by up to 30% FGT (Spear et al., 2014). This suggests that, rather than metabolizing (van de Wijgert et al., 2013; Vodstrcil et al., 2013). As many of the glycogen, certain bacterial species in the VMB might directly use the prior studies did not consider the specific method of (hormonal) simple sugars as substrates. Nevertheless, high free glycogen is contraception (i.e. grouping all contraception or all progestins) or associated with a Lactobacillus-dominant VMB (Mirmonsef et al., the duration of hormonal contraceptive treatment as independent 2014), and a clear relationship exists between estradiol and vaginal variables, and did not account for the potential confounding effect lactobacilli in post-menopausal women on HRT (Brotman et al., of including women with BV, the true effect of hormonal 2014b; Shen et al., 2016), albeit via incompletely understood contraceptives on the VMB has been difficult to discern. To mechanisms. address this, our group is conducting 16S rRNA gene sequencing in Although endogenous hormones can shape the VMB, we are a comprehensive cross-sectional clinical study where we control for only beginning to explore the modulation of the vaginal bacteria by many potential confounders. We aimed to examine the effect of hormonal contraceptives (Achilles and Hillier, 2013; Brotman et al., hormonal contraceptives on VMB alpha-diversity (Box 1) and the 2014a). This is a timely and controversial topic, due to the potential vaginal microenvironment. We thought that this was particularly for hormone-VMB interactions to manipulate HIV-1 target cells in warranted given recent publications, which demonstrated that the reproductive tract mucosa. Meta-analyses show that women on women with non-Lactobacillus-dominant VMB were at greater

DMPA are 40% more likely to acquire HIV-1 than women that do risk of HIV-1 infection (Gosmann et al., 2017), and which identified Disease Models & Mechanisms

9 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147 hormonal contraceptives and a diverse VMB as the strongest and thus enhance susceptibility to HIV-1 infection. A summary of predictors of genital inflammation (Lennard et al., 2017). Taken how DMPA might modulate the VMB and susceptibility to HIV-1 together, we propose that one of the mechanisms by which DMPA in women is presented in Fig. 4. enhances susceptibility to HIV-1 may result from its hypo- estrogenic effect. In the absence of estradiol, the vaginal Conclusions epithelium and/or mucus may become depleted of Lactobacillus- Herein, we have summarized the current literature and our view of promoting glycogen, which subsequently allows a diverse array of how the sex-hormone–microbiome–immunity axis has the potential bacteria to colonize. Consequently, this diverse microbiota might to affect HIV-1 susceptibility in women. Factors that enhance enhance mucosal inflammation and/or HIV-1 target cell activation, inflammation/inflammatory cytokines, and thus HIV-1 target cells,

A

Upper female genital tract Ovary Uterus Endocervix Lower Ectocervix female Vaginal tract genital tract

B C Vaginal Vaginal lumen lumen

Glycogen Glycogen Lactobacilli Lactobacilli Innate Innate inflammation Epithelium inflammation Activated T cells Activated T cells HIV susceptibility HIV susceptibility Stroma

Blood Blood vessel vessel

Estradiol MPA (hypo-estrogenic)

Key HIV-1 Lactobacilli Cytokines AMPs Glycogen

Diverse microbiota Dendritic cell Mucins T cell Activated T cell

Macrophage

Fig. 4. The sex-hormone–microbiome–immunity axis and HIV-1 susceptibility in women. An illustration depicting how the hormonal milieu of the lower female genital tract (FGT) might impact susceptibility to HIV-1. (A) Anatomy of the FGT. (B,C) Immunity and the microbiota in the lower FGT. (B) When estradiol is dominant, glycogen, a glucose polysaccharide that is correlated with enhanced vaginal lactobacilli, is abundant and adequately sustains the colonization of the vaginal mucosa by Lactobacillus species, via unknown mechanisms. Several studies have reported that a vaginal microbiota (VMB) dominated by species of lactobacilli is protective against HIV-1, perhaps a result of dampened innate inflammation and a reduction in activated T cells. In a microenvironment influenced by estradiol, epidemiological and experimental studies collectively suggest that susceptibility to HIV-1 appears to be hampered (Hapgood et al., 2018). (C) Conversely, when DMPA (MPA is the active ingredient in DMPA) is dominant, endogenous estradiol is suppressed due to the hypo-estrogenic effect of DMPA. As a result, glycogen deposition in the vaginal epithelium may be minimized, depleting one of the important nutrients that may sustain the protective vaginal lactobacilli. Given the depletion in nutrients, lactobacilli are not as numerous as when they are under the influence of estradiol, and a more diverse array of bacteria is subsequently supported in the VMB. In turn, the diverse bacteria might induce innate immunity in the FGT, upregulating cytokines, inflammation and activated T cells. Given that activated T cells are a major target of HIV-1, this type of environment would ultimately enhance susceptibility to HIV-1 in women. At present, it is unclear whether this proposed mechanism is a direct result of exposure to DMPA or an indirect result of hypo-estrogenism. AMPs, antimicrobial peptides; DMPA, depot-medroxyprogesterone acetate; HIV-1, human immunodeficiency virus type 1; MPA,

medroxyprogesterone acetate. Disease Models & Mechanisms

10 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147 in the FGT can modify the risk of infection. Factors that affect the Arnold, K. B., Burgener, A., Birse, K., Romas, L., Dunphy, L. J., Shahabi, K., integrity of the protective epithelial barrier in the FGT can also Abou, M., Westmacott, G. R., McCorrister, S., Kwatampora, J. et al. (2016). Increased levels of inflammatory cytokines in the female reproductive tract are modify the risk of HIV-1 infection by allowing viral particles to associated with altered expression of proteases, mucosal barrier proteins and an access target cells more readily. Although a relatively recent finding, influx of HIV-susceptible target cells. Mucosal Immunol. 9, 194-205. factors that modify the vaginal microbiota are also believed to be Arranz, E., Peña, A. S. and Bernardo, D. (2013). Mediators of inflammation and immune responses in the human . Mediat. Inflamm. 2013, able to modify HIV-1 risk via their impact on both inflammation 865638. and barrier integrity. Moving forward, rigorous clinical and Atashili, J., Poole, C., Ndumbe, P. M., Adimora, A. A. and Smith, J. S. (2008). preclinical studies aimed at examining how hormonal Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies. contraceptives affect the vaginal microbiota are critically needed. AIDS 22, 1493-1501. Ballweber, L., Robinson, B., Kreger, A., Fialkow, M., Lentz, G., McElrath, M. J. Understanding the factors that influence VMB composition, and the and Hladik, F. (2011). Vaginal langerhans cells nonproductively transporting HIV- mechanisms by which these bacteria modify host factors, including 1 mediate infection of T cells. J. Virol. 85, 13443-13447. the population of HIV-1 target cells, may help in understanding why Birse, K., Arnold, K. B., Novak, R. M., McCorrister, S., Shaw, S., Westmacott, women using DMPA as a contraceptive are up to 40% more likely to G. R., Ball, T. B., Lauffenburger, D. A. and Burgener, A. (2015a). Molecular signatures of immune activation and epithelial barrier remodeling are enhanced acquire HIV-1 than women not using hormonal contraceptives during the luteal phase of the menstrual cycle: implications for HIV susceptibility. (Gosmann et al., 2017). J. Virol. 89, 8793-8805. Birse, K. D. M., Cole, A. L., Hirbod, T., McKinnon, L., Ball, T. B., Westmacott, Acknowledgements G. R., Kimani, J., Plummer, F., Cole, A. M., Burgener, A. et al. (2015b). Non- The authors thank and acknowledge the contributions of members of the Kaushic cationic proteins are associated with HIV neutralizing activity in genital secretions lab over the years who have contributed to the papers cited in this Review. The of female sex workers. PLoS ONE 10, e0130404. Birse, K. D., Romas, L. M., Guthrie, B. L., Nilsson, P., Bosire, R., Kiarie, J., authors also acknowledge the artwork of Medical Illustrator Nancy Chu Ji in Fig. 4 Farquhar, C., Broliden, K. and Burgener, A. D. (2017). Genital injury signatures and for elements of Fig. 1. The authors acknowledge Philip Nguyen for the original and microbiome alterations associated with depot medroxyprogesterone acetate design of Fig.1. usage and intravaginal drying practices. J. Infect. Dis. 215, 590-598. Borgdorff, H., Verwijs, M. C., Wit, F. W. N. M., Tsivtsivadze, E., Ndayisaba, G. F., Competing interests Verhelst, R., Schuren, F. H. and van de Wijgert, J. H. H. M. (2015). The impact The authors declare no competing or financial interests. of hormonal contraception and pregnancy on sexually transmitted infections and on cervicovaginal microbiota in african sex workers. Sex. Transm. Dis. 42, Funding 143-152. This work was supported by the Canadian Institutes of Health Research [CIHR Borgdorff, H., Gautam, R., Armstrong, S. D., Xia, D., Ndayisaba, G. F., van Operating Grant FRN#126019 to C.K.]; the Canadian Institutes of Health Research Teijlingen, N. H., Geijtenbeek, T. B. H., Wastling, J. M. and van de Wijgert, Team Grant on Mucosal Immunology of HIV Vaccine Development [FRN#138657 to J. H. H. M. (2016). Cervicovaginal microbiome dysbiosis is associated with C.K.]; the Ontario HIV Treatment Network (OHTN) Applied HIV Research Chair [to proteome changes related to alterations of the cervicovaginal mucosal barrier. C.K.]; the Ontario Women’s Health Scholars Award from the Council of Ontario Mucosal Immunol. 9, 621-633. Universities (COU) [to J.M.W.]; and the Canadian Institutes of Health Research Bradshaw, C. S., Walker, S. M., Vodstrcil, L. A., Bilardi, J. E., Law, M., Hocking, Fellowship Award [to J.M.W.]. J. S., Fethers, K. A., Fehler, G., Petersen, S., Tabrizi, S. N. et al. (2014). The influence of behaviors and relationships on the vaginal microbiota of women and their female partners: the WOW health study. J. Infect. Dis. 209, 1562-1572. Brooks, J. P., Edwards, D. J., Blithe, D. L., Fettweis, J. M., Serrano, M. G., Sheth, References N. U., Strauss, J. F., Buck, G. A. and Jefferson, K. K. (2017). Effects of Abbai, N. S., Wand, H. and Ramjee, G. (2016). Biological factors that place women combined oral contraceptives, depot medroxyprogesterone acetate and the at risk for HIV: evidence from a large-scale clinical trial in Durban. BMC Womens levonorgestrel-releasing intrauterine system on the vaginal microbiome. Health 16, 19-19. Contraception 95, 405-413. Aboud, L., Ball, T. B., Tjernlund, A. and Burgener, A. (2014). The role of serpin Brotman, R. M., Ghanem, K. G., Klebanoff, M. A., Taha, T. E., Scharfstein, D. O. and cystatin antiproteases in mucosal innate immunity and their defense against and Zenilman, J. M. (2008). The effect of vaginal douching cessation on bacterial HIV. Am. J. Reprod. Immunol. 71, 12-23. vaginosis: a pilot study. Am. J. Obstet. Gynecol. 198, 628.e1-7. Achilles, S. L. and Hillier, S. L. (2013). The complexity of contraceptives: Brotman, R. M., Ravel, J., Bavoil, P. M., Gravitt, P. E. and Ghanem, K. G. (2014a). understanding their impact on genital immune cells and vaginal microbiota. AIDS Microbiome, sex hormones, and immune responses in the reproductive tract: 27 Suppl. 1, S5-S15. Challenges for vaccine development against sexually transmitted infections. Achilles, S. L., Creinin, M. D., Stoner, K. A., Chen, B. A., Meyn, L. and Hillier, Vaccine 32, 1543-1552. S. L. (2014). Changes in genital tract immune cell populations after initiation of Brotman, R. M., Shardell, M. D., Gajer, P., Fadrosh, D., Chang, K., Silver, M. I., intrauterine contraception. Am. J. Obstet. Gynecol. 211, 489.e1-9. Viscidi, R. P., Burke, A. E., Ravel, J. and Gravitt, P. E. (2014b). Association Achilles, S. L., Austin, M. N., Meyn, L. A., Mhlanga, F., Chirenje, Z. M. and Hillier, between the vaginal microbiota, menopause status, and signs of vulvovaginal S. L. (2018). Impact of contraceptive initiation on vaginal microbiota. atrophy. Menopause 21, 450-458. Am. J. Obstet. Gynecol. 218, 622.e1-622.e10. Byrne, E. H., Anahtar, M. N., Cohen, K. E., Moodley, A., Padavattan, N., Ismail, Alvarez-Olmos, M. I., Barousse, M. M., Rajan, L., Van Der Pol, B. J., Fortenberry, N., Bowman, B. A., Olson, G. S., Mabhula, A., Leslie, A. et al. (2016). D., Orr, D. and Fidel, P. L. (2004). Vaginal lactobacilli in adolescents: presence Association between injectable progestin-only contraceptives and HIV acquisition and relationship to local and systemic immunity, and to bacterial vaginosis. Sex. and HIV target cell frequency in the female genital tract in South African women: a Transm. Dis. 31, 393-400. prospective cohort study. Lancet. Infect. Dis. 16, 441-448. Amsel, R., Totten, P. A., Spiegel, C. A., Chen, K. C., Eschenbach, D. and Carias, A. M., McCoombe, S., McRaven, M., Anderson, M., Galloway, N., Holmes, K. K. (1983). Nonspecific vaginitis. Diagnostic criteria and microbial and Vandergrift, N., Fought, A. J., Lurain, J., Duplantis, M., Veazey, R. S. et al. epidemiologic associations. Am. J. Med. 74, 14-22. (2013). Defining the interaction of HIV-1 with the mucosal barriers of the female Anahtar, M. N., Byrne, E. H., Doherty, K. E., Bowman, B. A., Yamamoto, H. S., reproductive tract. J. Virol. 87, 11388-11400. Soumillon, M., Padavattan, N., Ismail, N., Moodley, A., Sabatini, M. E. et al. Chaban, B., Links, M. G., Jayaprakash, T., Wagner, E. C., Bourque, D. K., Lohn, (2015). Cervicovaginal bacteria are a major modulator of host inflammatory Z., Albert, A. Y. K., van Schalkwyk, J., Reid, G., Hemmingsen, S. M. et al. responses in the female genital tract. Immunity 42, 965-976. (2014). Characterization of the vaginal microbiota of healthy Canadian women Anahtar, M. N., Gootenberg, D. B., Mitchell, C. M. and Kwon, D. S. (2018). through the menstrual cycle. Microbiome 2, 23-23. Cervicovaginal microbiota and reproductive health: the virtue of simplicity. Cell Chandra, N., Thurman, A. R., Anderson, S., Cunningham, T. D., Yousefieh, N., Host Microbe 23, 159-168. Mauck, C. and Doncel, G. F. (2013). Depot medroxyprogesterone acetate Anderson, D. J., Marathe, J. and Pudney, J. (2014). The structure of the human increases immune cell numbers and activation markers in human vaginal mucosal vaginal and its role in immune defense. Am. J. Reprod. Immunol. tissues. AIDS Res. Hum. Retrovir. 29, 592-601. 71, 618-623. Chappell, C. A., Isaacs, C. E., Xu, W., Meyn, L. A., Uranker, K., Dezzutti, C. S., Anipindi, V. C., Bagri, P., Roth, K., Dizzell, S. E., Nguyen, P. V., Shaler, C. R., Moncla, B. J. and Hillier, S. L. (2015). The effect of menopause on the innate Chu, D. K., Jiménez-Saiz, R., Liang, H., Swift, S. et al. (2016). Estradiol antiviral activity of cervicovaginal lavage. Am. J. Obstet. Gynecol. 213, 204.e1-6. enhances CD4+ T-cell anti-viral immunity by priming vaginal DCs to Induce Th17 Chen, R.-Y., Fan, Y.-M., Zhang, Q., Liu, S., Li, Q., Ke, G.-L., Li, C. and You, Z.

responses via an IL-1-dependent pathway. PLoS Pathog. 12, e1005589. (2015). Estradiol inhibits Th17 cell differentiation through inhibition of RORγT Disease Models & Mechanisms

11 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

transcription by recruiting the ERα/REA complex to estrogen response elements Forcey, D. S., Vodstrcil, L. A., Hocking, J. S., Fairley, C. K., Law, M., McNair, R. P. of the RORγT promoter. J. Immunol. 194, 4019-4028. and Bradshaw, C. S. (2015). Factors associated with bacterial vaginosis among Chen, C., Song, X., Wei, W., Zhong, H., Dai, J., Lan, Z., Li, F., Yu, X., Feng, Q., women who have sex with women: a systematic review. PLoS ONE 10, e0141905. Wang, Z. et al. (2017). The microbiota continuum along the female reproductive Franasiak, J. M., Werner, M. D., Juneau, C. R., Tao, X., Landis, J., Zhan, Y., Treff, tract and its relation to uterine-related diseases. Nat. Commun. 8, 875. N. R. and Scott, R. T. (2016). Endometrial microbiome at the time of embryo Christodoulides, M., Everson, J. S., Liu, B. L., Lambden, P. R., Watt, P. J., transfer: next-generation sequencing of the 16S ribosomal subunit. J. Assist. Thomas, E. J. and Heckels, J. E. (2000). Interaction of primary human Reprod. Genet. 33, 129-136. endometrial cells with Neisseria gonorrhoeae expressing green fluorescent Franklin, R. D. and Kutteh, W. H. (1999). Characterization of immunoglobulins and protein. Mol. Microbiol. 35, 32-43. cytokines in human cervical mucus: influence of exogenous and endogenous Cicala, C., Martinelli, E., McNally, J. P., Goode, D. J., Gopaul, R., Hiatt, J., hormones. J. Reprod. Immunol. 42, 93-106. Jelicic, K., Kottilil, S., Macleod, K., O’Shea, A. et al. (2009). The integrin Freeman, E. E., Weiss, H. A., Glynn, J. R., Cross, P. L., Whitworth, J. A. and alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset Hayes, R. J. (2006). Herpes simplex virus 2 infection increases HIV acquisition in that is highly susceptible to infection by HIV-1. Proc. Natl. Acad. Sci. USA 106, men and women: systematic review and meta-analysis of longitudinal studies. 20877-20882. AIDS 20, 73-83. Cruickshank, R. and Sharman, A. (1934). The biology of the vagina in the human Gajer, P., Brotman, R. M., Bai, G., Sakamoto, J., Schutte, U. M. E., Zhong, X., subject. BJOG 41, 208-226. Koenig, S. S. K., Fu, L., Ma, Z., Zhou, X. et al. (2012). Temporal dynamics of the De Seta, F., Restaino, S., De Santo, D., Stabile, G., Banco, R., Busetti, M., human vaginal microbiota. Sci. Transl. Med. 4, 132ra52. Barbati, G. and Guaschino, S. (2012). Effects of hormonal contraception on Geijtenbeek, T. B. H. and van Kooyk, Y. (2003). DC-SIGN: a novel HIV receptor on . Contraception 86, 526-529. DCs that mediates HIV-1 transmission. Curr. Top. Microbiol. Immunol. 276, 31-54. Doerflinger, S. Y., Throop, A. L. and Herbst-Kralovetz, M. M. (2014). Bacteria in Geijtenbeek, T. B. H., Kwon, D. S., Torensma, R., van Vliet, S. J., van the vaginal microbiome alter the innate immune response and barrier properties of Duijnhoven, G. C. F., Middel, J., Cornelissen, I. L. M. H. A., Nottet, H. S. L. M., the human vaginal epithelia in a species-specific manner. J. Infect. Dis. 209, KewalRamani, V. N., Littman, D. R. et al. (2000). DC-SIGN, a dendritic cell- 1989-1999. specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, Dunbar, B., Patel, M., Fahey, J. and Wira, C. (2012). Endocrine control of mucosal 587-597. ̈ immunity in the female reproductive tract: impact of environmental disruptors. Mol. Gerstner, G. J., Grunberger, W., Boschitsch, E. and Rotter, M. (1982). Vaginal Cell. Endocrinol. 354, 85-93. organisms in prepubertal children with and without vulvovaginitis. AVaginoscopic Enomoto, L. M., Kloberdanz, K. J., Mack, D. G., Elizabeth, D. and Weinberg, A. study. Arch. Gynecol. 231, 247-252. (2007). Ex vivo effect of estrogen and progesterone compared with Ghisletti, S., Meda, C., Maggi, A. and Vegeto, E. (2005). 17beta-estradiol inhibits dexamethasone on cell-mediated immunity of HIV-infected and uninfected inflammatory gene expression by controlling NF-kappaB intracellular localization. subjects. J. Acquired Immune Deficiency Syndromes 45, 137-143. Mol. Cell. Biol. 25, 2957-2968. Eschenbach, D. A., Rosene, K., Tompkins, L. S., Watkins, H. and Gravett, M. G. Ghosh, M., Shen, Z., Fahey, J. V., Cu-Uvin, S., Mayer, K. and Wira, C. R. (2010). (1986). Endometrial cultures obtained by a triple-lumen method from afebrile and Trappin-2/Elafin: a novel innate anti-human immunodeficiency virus-1 molecule of febrile postpartum women. J. Infect. Dis. 153, 1038-1045. the human female reproductive tract. Immunology 129, 207-219. Eschenbach, D. A., Thwin, S. S., Patton, D. L., Hooton, T. M., Stapleton, A. E., Ghosh, M., Shen, Z., Fahey, J. V., Crist, S. G., Patel, M., Smith, J. M. and Wira, C. R. (2013). Pathogen recognition in the human female reproductive tract: Agnew, K., Winter, C., Meier, A. and Stamm, W. E. (2000). Influence of the expression of intracellular cytosolic sensors NOD1, NOD2, RIG-1, and MDA5 and normal menstrual cycle on vaginal tissue, discharge, and microflora. Clin. Infect. response to HIV-1 and Neisseria gonorrhea. Am. J. Reprod. Immunol. 69, 41-51. Dis. 30, 901-907. Ginkel, P. D., Soper, D. E., Bump, R. C. and Dalton, H. P. (1993). Vaginal flora in Eslahpazir, J., Jenabian, M.-A., Bouhlal, H., Hocini, H., Carbonneil, C., postmenopausal women: the effect of estrogen replacement. Infect. Dis. Obstet. Grésenguet, G., Kéou, F.-X. M., LeGoff, J., Saïdi, H., Requena, M. et al. Gynecol. 1, 94-97. (2008). Infection of macrophages and dendritic cells with primary R5-tropic human Gipson, I. K., Ho, S. B., Spurr-Michaud, S. J., Tisdale, A. S., Zhan, Q., immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 Torlakovic, E., Pudney, J., Anderson, D. J., Toribara, N. W. and Hill, J. A. antibodies purified from cervicovaginal secretions. Clin. Vaccine Immunol. 15, (1997). Mucin genes expressed by human female reproductive tract epithelia. 872-884. Biol. Reprod. 56, 999-1011. Fahey, J. V., Schaefer, T. M., Channon, J. Y. and Wira, C. R. (2005). Secretion of Givan, A. L., White, H. D., Stern, J. E., Colby, E., Gosselin, E. J., Guyre, P. M. and cytokines and chemokines by polarized human epithelial cells from the female Wira, C. R. (1997). Flow cytometric analysis of leukocytes in the human female reproductive tract. Hum. Reprod. 20, 1439-1446. reproductive tract: comparison of fallopian tube, uterus, cervix, and vagina. Fahey, J. V., Wright, J. A., Shen, L., Smith, J. M., Ghosh, M., Rossoll, R. M. and Am. J. Reprod. Immunol. 38, 350-359. Wira, C. R. (2008). Estradiol selectively regulates innate immune function by Gosmann, C., Anahtar, M. N., Handley, S. A., Farcasanu, M., Abu-Ali, G., polarized human uterine epithelial cells in culture. Mucosal Immunol. 1, 317-325. Bowman, B. A., Padavattan, N., Desai, C., Droit, L., Moodley, A. et al. (2017). Farage, M. and Maibach, H. (2006). Lifetime changes in the and vagina. Arch. Lactobacillus-deficient cervicovaginal bacterial communities are associated with Gynecol. Obstet. 273, 195-202. increased HIV acquisition in young south african women. Immunity 46, 29-37. Ferreira, V. H., Nazli, A., Khan, G., Mian, M. F., Ashkar, A. A., Gray-Owen, S., Gregoire, A. T. and Parakkal, P. F. (1972). Glycogen content in the vaginal tissue of Kaul, R. and Kaushic, C. (2011). Endometrial epithelial cell responses to normally cycling and estrogen and progesterone-treated rhesus monkeys. Biol. coinfecting viral and bacterial pathogens in the genital tract can activate the HIV-1 Reprod. 7, 9-14. LTR in an NF{kappa}B-and AP-1-dependent manner. J. Infect. Dis. 204, 299-308. Gregoire, A. T. and Richardson, D. W. (1970). Glycogen and water responses to Ferreira, V. H., Nazli, A., Mossman, K. L. and Kaushic, C. (2013). Proinflammatory estrogen in the hamster reproductive tract. Endocrinology 87, 1369-1372. β cytokines and chemokines - but not interferon- - produced in response to HSV-2 Hapgood, J. P., Kaushic, C. and Hel, Z. (2018). Hormonal contraception and HIV-1 in primary human genital epithelial cells are associated with viral replication and acquisition: biological mechanisms. Endocr. Rev. 39, 36-78. the presence of the Virion host shutoff protein. Am. J. Reprod. Immunol. 70, Hardy, L., Jespers, V., De Baetselier, I., Buyze, J., Mwambarangwe, L., 199-212. Musengamana, V., van de Wijgert, J. and Crucitti, T. (2017). Association of Ferreira, V. H., Kafka, J. K. and Kaushic, C. (2014). Influence of common mucosal vaginal dysbiosis and biofilm with contraceptive vaginal ring biomass in African co-factors on HIV infection in the female genital tract. Am. J. Reprod. Immunol. 71, women. PLoS ONE 12, e0178324. 543-554. Hart, K. M., Murphy, A. J., Barrett, K. T., Wira, C. R., Guyre, P. M. and Pioli, P. A. Ferreira, V. H., Nazli, A., Dizzell, S. E., Mueller, K. and Kaushic, C. (2015). The (2009). Functional expression of pattern recognition receptors in tissues of the anti-inflammatory activity of curcumin protects the genital mucosal epithelial human female reproductive tract. J. Reprod. Immunol. 80, 33-40. barrier from disruption and blocks replication of HIV-1 and HSV-2. PLoS ONE 10, Hemsell, D. L., Obregon, V. L., Heard, M. C. and Nobles, B. J. (1989). Endometrial e0124903. bacteria in asymptomatic, nonpregnant women. J. Reprod. Med. 34, 872-874. Fichorova, R. N., Cronin, A. O., Lien, E., Anderson, D. J. and Ingalls, R. R. Herbst-Kralovetz, M. M., Quayle, A. J., Ficarra, M., Greene, S., Rose, W. A., (2002). Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells Chesson, R., Spagnuolo, R. A. and Pyles, R. B. (2008). Quantification and occurs in the absence of toll-like receptor 4-mediated signaling. J. Immunol. 168, comparison of toll-like receptor expression and responsiveness in primary and 2424-2432. immortalized human female lower genital tract epithelia. Am. J. Reprod. Immunol. Fichorova, R. N., Yamamoto, H. S., Delaney, M. L., Onderdonk, A. B. and 59, 212-224. Doncel, G. F. (2011). Novel vaginal microflora colonization model providing new Hickey, D. K., Patel, M. V., Fahey, J. V. and Wira, C. R. (2011). Innate and adaptive insight into microbicide mechanism of action. mBio 2, e00168. immunity at mucosal surfaces of the female reproductive tract: stratification and Fichorova, R. N., Chen, P.-L., Morrison, C. S., Doncel, G. F., Mendonca, K., integration of immune protection against the transmission of sexually transmitted Kwok, C., Chipato, T., Salata, R. and Mauck, C. (2015). The contribution of infections. J. Reprod. Immunol. 88, 185-194. cervicovaginal infections to the immunomodulatory effects of hormonal Hickey, R. J., Zhou, X., Settles, M. L., Erb, J., Malone, K., Hansmann, M. A.,

contraception. mBio 6, e00221-15. Shew, M. L., Van Der Pol, B., Fortenberry, J. D. and Forney, L. J. (2015). Disease Models & Mechanisms

12 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Vaginal microbiota of adolescent girls prior to the onset of menarche resemble Marrazzo, J. M., Koutsky, L. A., Eschenbach, D. A., Agnew, K., Stine, K. and those of reproductive-age women. mBio 6, e00097-15. Hillier, S. L. (2002). Characterization of vaginal flora and bacterial vaginosis in Hill, G. B., St Claire, K. K. and Gutman, L. T. (1995). Anaerobes predominate women who have sex with women. J. Infect. Dis. 185, 1307-1313. among the vaginal microflora of prepubertal girls. Clin. Infect. Dis. 20 Suppl. 2, Martın,́ R., Soberón, N., Vaneechoutte, M., Flórez, A. B., Vázquez, F. and S269-S270. Suárez, J. E. (2008). Characterization of indigenous vaginal lactobacilli from Hladik, F. and McElrath, M. J. (2008). Setting the stage: host invasion by HIV. Nat. healthy women as probiotic candidates. Int. Microbiol. 11, 261-266. Rev. Immunol. 8, 447-457. Masson, L., Passmore, J.-A. S., Liebenberg, L. J., Werner, L., Baxter, C., Hocini, H., Becquart, P., Bouhlal, H., Chomont, N., Ancuta, P., Kazatchkine, Arnold, K. B., Williamson, C., Little, F., Mansoor, L. E., Naranbhai, V. et al. M. D. and Bélec, L. (2001). Active and selective transcytosis of cell-free human (2015). Genital inflammation and the risk of HIV acquisition in women. Clin. Infect. immunodeficiency virus through a tight polarized monolayer of human Dis. 61, 260-269. endometrial cells. J. Virol. 75, 5370-5374. McKinnon, L. R., Nyanga, B., Chege, D., Izulla, P., Kimani, M., Huibner, S., Houlihan, C. F., Larke, N. L., Watson-Jones, D., Smith-McCune, K. K., Shiboski, Gelmon, L., Block, K. E., Cicala, C., Anzala, A. O. et al. (2011). Characterization S., Gravitt, P. E., Smith, J. S., Kuhn, L., Wang, C. and Hayes, R. (2012). Human of a human cervical CD4+ T cell subset coexpressing multiple markers of HIV papillomavirus infection and increased risk of HIV acquisition. A systematic review susceptibility. J. Immunol. 187, 6032-6042. and meta-analysis. AIDS 26, 2211-2222. McKinnon, L. R., Nyanga, B., Kim, C. J., Izulla, P., Kwatampora, J., Kimani, M., Huang, Y., Merkatz, R. B., Hillier, S. L., Roberts, K., Blithe, D. L., Sitruk-Ware, R. Shahabi, K., Mugo, N., Smith, J. S., Anzala, A. O. et al. (2015). Early HIV-1 and Creinin, M. D. (2015). Effects of a one year reusable contraceptive vaginal infection is associated with reduced frequencies of cervical Th17 cells. JAIDS ring on vaginal microflora and the risk of vaginal infection: an open-label J. Acquir. Immune Defic. Syndr. 68, 6-12. prospective evaluation. PLoS ONE 10, e0134460. McNeely, T. B., Dealy, M., Dripps, D. J., Orenstein, J. M., Eisenberg, S. P. and Huijbregts, R. P. H., Michel, K. G. and Hel, Z. (2014). Effect of progestins on Wahl, S. M. (1995). Secretory leukocyte protease inhibitor: a human saliva protein immunity: medroxyprogesterone but not norethisterone or levonorgestrel exhibiting anti-human immunodeficiency virus 1 activity in vitro. J. Clin. Invest. 96, suppresses the function of T cells and pDCs. Contraception 90, 123-129. 456-464. Jacobs, J. and Braun, J. (2014). Host genes and their effect on the intestinal Michel, K. G., Huijbregts, R. P. H., Gleason, J. L., Richter, H. E. and Hel, Z. microbiome garden. Genome Med. 6, 119. (2015). Effect of hormonal contraception on the function of plasmacytoid dendritic Jespers, V., Kyongo, J., Joseph, S., Hardy, L., Cools, P., Crucitti, T., Mwaura, cells and distribution of immune cell populations in the female reproductive tract. M., Ndayisaba, G., Delany-Moretlwe, S., Buyze, J. et al. (2017). A longitudinal JAIDS J. Acquir. Immune Defic. Syndr. 68, 511-518. analysis of the vaginal microbiota and vaginal immune mediators in women from Miller, C. J. and Shattock, R. J. (2003). Target cells in vaginal HIV transmission. sub-Saharan Africa. Sci. Rep. 7, 11974. Microbes Infect. 5, 59-67. Kaushic, C. (2009). The role of the local microenvironment in regulating Mirmonsef, P. and Spear, G. T. (2014). The barrier to HIV transmission provided by susceptibility and immune responses to sexually transmitted viruses in the genital tract lactobacillus colonization. Am. J. Reprod. Immunol. 71, 531-536. female genital tract. J. Reprod. Immunol. 83, 168-172. Mirmonsef, P., Hotton, A. L., Gilbert, D., Burgad, D., Landay, A., Weber, K. M., Kaushic, C. (2011). HIV-1 infection in the female reproductive tract: role of Cohen, M., Ravel, J. and Spear, G. T. (2014). Free glycogen in vaginal fluids is interactions between HIV-1 and genital epithelial cells. Am. J. Reprod. Immunol. associated with lactobacillus colonization and low vaginal pH. PLoS ONE 9, 65, 253-260. e102467-e102467. Kaushic, C., Roth, K. L., Anipindi, V. and Xiu, F. (2011). Increased prevalence of Mirmonsef, P., Hotton, A. L., Gilbert, D., Gioia, C. J., Maric, D., Hope, T. J., sexually transmitted viral infections in women: the role of female sex hormones in Landay, A. L. and Spear, G. T. (2016). Glycogen levels in undiluted genital fluid regulating susceptibility and immune responses. J. Reprod. Immunol. 88, and their relationship to vaginal pH, estrogen, and progesterone. PLoS ONE 11, 204-209. e0153553. Kazi, Y. F., Saleem, S. and Kazi, N. (2012). Investigation of vaginal microbiota in Mitchell, C. M., McLemore, L., Westerberg, K., Astronomo, R., Smythe, K., sexually active women using hormonal contraceptives in Pakistan. BMC Urol. 12, Gardella, C., Mack, M., Magaret, A., Patton, D., Agnew, K. et al. (2014). Long- 22. term effect of depot medroxyprogesterone acetate on vaginal microbiota, Kinross, J. M., Darzi, A. W. and Nicholson, J. K. (2011). Gut microbiome-host epithelial thickness and HIV target cells. J. Infect. Dis. 210, 651-655. interactions in health and disease. Genome Med. 3, 14-14. Mitchell, C. M., Haick, A., Nkwopara, E., Garcia, R., Rendi, M., Agnew, K., Kumamoto, Y. and Iwasaki, A. (2012). Unique features of antiviral immune system Fredricks, D. N. and Eschenbach, D. (2015). Colonization of the upper genital of the vaginal mucosa. Curr. Opin. Immunol. 24, 411-416. tract by vaginal bacterial species in nonpregnant women. Am. J. Obstet. Gynecol. Kutteh, W. H., Moldoveanu, Z. and Mestecky, J. (1998). Mucosal immunity in the 212, 611.e1-611.e9. female reproductive tract: correlation of immunoglobulins, cytokines, and Møller, B. R., Kristiansen, F. V., Thorsen, P., Frost, L. and Mogensen, S. C. reproductive hormones in human cervical mucus around the time of ovulation. (1995). Sterility of the . Acta Obstet. Gynecol. Scand. 74, 216-219. AIDS Res. Hum. Retrovir. 14 Suppl. 1, S51-S55. Moreno, I., Codoñer, F. M., Vilella, F., Valbuena, D., Martinez-Blanch, J. F., Lai, S. K., Hida, K., Shukair, S., Wang, Y.-Y., Figueiredo, A., Cone, R., Hope, T. J. Jimenez-Almazán, J., Alonso, R., Alamá, P., Remohı,́ J., Pellicer, A. et al. and Hanes, J. (2009). Human immunodeficiency virus type 1 is trapped by acidic (2016). Evidence that the endometrial microbiota has an effect on implantation but not by neutralized human cervicovaginal mucus. J. Virol. 83, 11196-11200. success or failure. Am. J. Obstet. Gynecol. 215, 684-703. Lajoie, J., Juno, J., Burgener, A., Rahman, S., Mogk, K., Wachihi, C., Mwanjewe, Morrison, C. S., Chen, P.-L., Kwok, C., Baeten, J. M., Brown, J., Crook, A. M., J., Plummer, F. A., Kimani, J., Ball, T. B. et al. (2012). A distinct cytokine and Van Damme, L., Delany-Moretlwe, S., Francis, S. C., Friedland, B. A. et al. chemokine profile at the genital mucosa is associated with HIV-1 protection (2015). Hormonal contraception and the risk of HIV acquisition: an individual among HIV-exposed seronegative commercial sex workers. Mucosal Immunol. 5, participant data meta-analysis. PLoS Med. 12, e1001778-e1001778. 277-287. Muzny, C. A. and Schwebke, J. R. (2016). Pathogenesis of Bacterial Vaginosis: Łaniewski, P., Gomez, A., Hire, G., So, M. and Herbst-Kralovetz, M. M. (2017). Discussion of Current Hypotheses. J. Infect. Dis. 214, S1-S5. Human three-dimensional endometrial epithelial cell model to study host Nazli, A., Yao, X.-D., Smieja, M., Rosenthal, K. L., Ashkar, A. A. and Kaushic, C. interactions with vaginal bacteria and Neisseria gonorrhoeae. Infect. Immun. (2009). Differential induction of innate anti-viral responses by TLR ligands against 85, e01049-e01016. Herpes simplex virus, type 2, infection in primary genital epithelium of women. Lee, S. K., Kim, C. J., Kim, D.-J. and Kang, J.-H. (2015). Immune cells in the Antivir. Res. 81, 103-112. female reproductive tract. Immune Network 15, 16-26. Nazli, A., Chan, O., Dobson-Belaire, W. N., Ouellet, M., Tremblay, M. J., Gray- Lennard, K., Dabee, S., Barnabas, S. L., Havyarimana, E., Blakney, A., Owen, S. D., Arsenault, A. L. and Kaushic, C. (2010). Exposure to HIV-1 directly Jaumdally, S. Z., Botha, G., Mkhize, N. N., Bekker, L.-G., Lewis, D. A. et al. impairs mucosal epithelial barrier integrity allowing microbial translocation. PLoS (2017). Microbial composition predicts genital tract inflammation and persistent Pathog. 6, e1000852. bacterial vaginosis in adolescent South African women. Infect. Immun. 86, Nazli, A., Kafka, J. K., Ferreira, V. H., Anipindi, V., Mueller, K., Osborne, B. J., e00410-e00417. Dizzell, S., Chauvin, S., Mian, M. F., Ouellet, M. et al. (2013). HIV-1 gp120 Li, Z., Palaniyandi, S., Zeng, R., Tuo, W., Roopenian, D. C. and Zhu, X. (2011). induces TLR2- and TLR4-mediated innate immune activation in human female Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc genital epithelium. J. Immunol. 191, 4246-4258. receptor (FcRn) confers protective immunity to vaginal infection. Proc. Natl. Acad. Nazli, A., Dizzell, S., Zahoor, M. A., Ferreira, V. H., Kafka, J., Woods, M. W., Sci. USA 108, 4388-4393. Ouellet, M., Ashkar, A. A., Tremblay, M. J., Bowdish, D. M. E. et al. (2018). Low, N., Chersich, M. F., Schmidlin, K., Egger, M., Francis, S. C., van de Wijgert, Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via J. H. H. M., Hayes, R. J., Baeten, J. M., Brown, J., Delany-Moretlwe, S. et al. Toll-like-receptor 2 pathway acts to protect the mucosal barrier. Cell. Mol. (2011). Intravaginal practices, bacterial vaginosis, and HIV infection in women: Immunol. individual participant data meta-analysis. PLoS Med. 8, e1000416. Neggers, Y. H., Nansel, T. R., Andrews, W. W., Schwebke, J. R., Yu, K.-F., Ma, B., Forney, L. J. and Ravel, J. (2012). Vaginal microbiome: rethinking health Goldenberg, R. L. and Klebanoff, M. A. (2007). Dietary intake of selected

and disease. Annu. Rev. Microbiol. 66, 371-389. nutrients affects bacterial vaginosis in women. J. Nutr. 137, 2128-2133. Disease Models & Mechanisms

13 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Nguyen, P. V., Kafka, J. K., Ferreira, V. H., Roth, K. and Kaushic, C. (2014). Innate S Lashkari, B. and Anumba, D. O. C. (2017). Estradiol alters the immune- and adaptive immune responses in male and female reproductive tracts in responsiveness of cervical epithelial cells stimulated with ligands of Toll-like homeostasis and following HIV infection. Cell. Mol. Immunol. 11, 410-427. receptors 2 and 4. PLoS ONE 12, e0173646. Nguyen, P. V., Wessels, J. M., Mueller, K., Vahedi, F., Anipindi, V., Verschoor, Saba, E., Origoni, M., Taccagni, G., Ferrari, D., Doglioni, C., Nava, A., Lisco, A., C. P., Chew, M., Deshiere, A., Karniychuk, U., Mazzulli, T. et al. (2017). Grivel, J.-C., Margolis, L. and Poli, G. (2013). Productive HIV-1 infection of Frequency of human CD45+ target cells is a key determinant of intravaginal HIV-1 human cervical tissue ex vivo is associated with the secretory phase of the infection in humanized mice. Sci. Rep. 7, 15263. menstrual cycle. Mucosal Immunol. 6, 1081-1090. Norvell, M. K., Benrubi, G. I. and Thompson, R. J. (1984). Investigation of Safaeian, M., Falk, R. T., Rodriguez, A. C., Hildesheim, A., Kemp, T., Williams, microtrauma after sexual intercourse. J. Reprod. Med. 29, 269-271. M., Morera, L., Barrantes, M., Herrero, R., Porras, C. et al. (2009). Factors Nunn, K. L. and Forney, L. J. (2016). Unraveling the dynamics of the human vaginal associated with fluctuations in IgA and IgG levels at the cervix during the microbiome. Yale J. Biol. Med. 89, 331-337. menstrual cycle. J. Infect. Dis. 199, 455-463. Nunn, K. L., Wang, Y.-Y., Harit, D., Humphrys, M. S., Ma, B., Cone, R., Ravel, J. Schaefer, T. M., Wright, J. A., Pioli, P. A. and Wira, C. R. (2005). IL-1beta- and Lai, S. K. (2015). Enhanced trapping of HIV-1 by human cervicovaginal mediated proinflammatory responses are inhibited by estradiol via down- mucus is associated with lactobacillus crispatus-dominant microbiota. mBio 6, regulation of IL-1 receptor type I in uterine epithelial cells. J. Immunol. 175, e01084-15. 6509-6516. Paavonen, J. (1983). Physiology and ecology of the vagina. Scand. J. Infect. Dis. Schwebke, J. R., Richey, C. M. and Weiss, H. L. (1999). Correlation of behaviors Suppl. 40, 31-35. with microbiological changes in vaginal flora. J. Infect. Dis. 180, 1632-1636. Patel, M. V., Fahey, J. V., Rossoll, R. M. and Wira, C. R. (2013). Innate immunity in Schwebke, J. R., Muzny, C. A. and Josey, W. E. (2014). Role of Gardnerella the vagina (part I): estradiol inhibits HBD2 and elafin secretion by human vaginal vaginalis in the pathogenesis of bacterial vaginosis: a conceptual model. J. Infect. epithelial cells. Am. J. Reprod. Immunol. 69, 463-474. Dis. 210, 338-343. Petrova, M. I., Lievens, E., Malik, S., Imholz, N. and Lebeer, S. (2015). Shen, R., Richter, H. E. and Smith, P. D. (2011). Early HIV-1 target cells in human Lactobacillus species as biomarkers and agents that can promote various vaginal and ectocervical mucosa. Am. J. Reprod. Immunol. 65, 261-267. aspects of vaginal health. Front. Physiol. 6, 81. Shen, J., Song, N., Williams, C. J., Brown, C. J., Yan, Z., Xu, C. and Forney, L. J. Piccinni, M. P., Giudizi, M. G., Biagiotti, R., Beloni, L., Giannarini, L., (2016). Effects of low dose estrogen therapy on the vaginal microbiomes of Sampognaro, S., Parronchi, P., Manetti, R., Annunziato, F. and Livi, C. women with . Sci. Rep. 6, 24380. (1995). Progesterone favors the development of human T helper cells producing Shen, Z., Rodriguez-Garcia, M., Ochsenbauer, C. and Wira, C. R. (2017). Th2-type cytokines and promotes both IL-4 production and membrane CD30 Characterization of immune cells and infection by HIV in human ovarian tissues. expression in established Th1 cell clones. J. Immunol. 155, 128-133. Am. J. Reprod. Immunol. 78, e12687-e12687. Pioli, P. A., Amiel, E., Schaefer, T. M., Connolly, J. E., Wira, C. R. and Guyre, Shukair, S. A., Allen, S. A., Cianci, G. C., Stieh, D. J., Anderson, M. R., Baig, P. M. (2004). Differential expression of toll-like receptors 2 and 4 in tissues of the S. M., Gioia, C. J., Spongberg, E. J., Kauffman, S. M., McRaven, M. D. et al. (2013). Human cervicovaginal mucus contains an activity that hinders HIV-1 human female reproductive tract. Infect. Immun. 72, 5799-5806. Polis, C. B., Curtis, K. M., Hannaford, P. C., Phillips, S. J., Chipato, T., Kiarie, movement. Mucosal Immunol. 6, 427-434. Shust, G. F., Cho, S., Kim, M., Madan, R. P., Guzman, E. M., Pollack, M., Epstein, J. N., Westreich, D. J. and Steyn, P. S. (2016). An updated systematic review of J., Cohen, H. W., Keller, M. J. and Herold, B. C. (2010). Female genital tract epidemiological evidence on hormonal contraceptive methods and HIV secretions inhibit herpes simplex virus infection: correlation with soluble mucosal acquisition in women. AIDS 30, 2665-2683. immune mediators and impact of hormonal contraception. Am. J. Reprod. Pope, M. and Haase, A. T. (2003). Transmission, acute HIV-1 infection and the Immunol. 63, 110-119. quest for strategies to prevent infection. Nat. Med. 9, 847-852. Silva, C., Rey, R. and Elena Nader-Macıas,́ M. (2004). Effects of Estrogen Prakash, M., Kapembwa, M. S., Gotch, F. and Patterson, S. (2002). Oral Administration on the Colonization Capability of Lactobacilli and Escherichia coli contraceptive use induces upregulation of the CCR5 chemokine receptor on in the Urinary Tracts of Mice, Vol. 268, pp. 387-400. New Jersey: Humana Press. CD4(+) T cells in the cervical epithelium of healthy women. J. Reprod. Immunol. Spear, G. T., French, A. L., Gilbert, D., Zariffard, M. R., Mirmonsef, P., Sullivan, 54, 117-131. T. H., Spear, W. W., Landay, A., Micci, S., Lee, B.-H. et al. (2014). Human α- Pudney, J., Quayle, A. J. and Anderson, D. J. (2005). Immunological amylase present in lower-genital-tract mucosal fluid processes glycogen to microenvironments in the human vagina and cervix: mediators of cellular support vaginal colonization by lactobacillus. J. Infect. Dis. 210, 1019-1028. immunity are concentrated in the cervical transformation zone. Biol. Reprod. 73, Stewart-Tull, D. E. S. (1964). Evidence that vaginal lactobacilli do not ferment 1253-1263. glycogen. Am. J. Obstet. Gynecol. 88, 676-679. Quayle, A. J. (2002). The innate and early immune response to pathogen challenge Stieh, D. J., Maric, D., Kelley, Z. L., Anderson, M. R., Hattaway, H. Z., Beilfuss, in the female genital tract and the pivotal role of epithelial cells. J. Reprod. B. A., Rothwangl, K. B., Veazey, R. S. and Hope, T. J. (2014). Vaginal challenge Immunol. 57, 61-79. with an SIV-based dual reporter system reveals that infection can occur Quispe Calla, N. E., Ghonime, M. G., Cherpes, T. L. and Vicetti Miguel, R. D. throughout the upper and lower female reproductive tract. PLoS Pathog. 10, (2015). Medroxyprogesterone acetate impairs human dendritic cell activation and e1004440. function. Hum. Reprod. 30, 1169-1177. Straub, R. H. (2007). The complex role of estrogens in inflammation. Endocr. Rev. Ravel, J., Gajer, P., Abdo, Z., Schneider, G. M., Koenig, S. S. K., McCulle, S. L., 28, 521-574. Karlebach, S., Gorle, R., Russell, J., Tacket, C. O. et al. (2011). Vaginal Sun, L., Finnegan, C. M., Kish-Catalone, T., Blumenthal, R., Garzino-Demo, P., microbiome of reproductive-age women. Proc. Natl. Acad. Sci. USA 108 Suppl., La Terra Maggiore, G. M., Berrone, S., Kleinman, C., Wu, Z., Abdelwahab, S. 4680-4687. et al. (2005). Human beta-defensins suppress human immunodeficiency virus Reed, B. G. and Carr, B. R. (2000). The Normal Menstrual Cycle and the Control of infection: potential role in mucosal protection. J. Virol. 79, 14318-14329. Ovulation: in www.endotext.org (ed. De Groot L. J., Chrousos G., Dungan K. Swidsinski, A., Loening-Baucke, V., Mendling, W., Dörffel, Y., Schilling, J., et al.). South Dartmouth: MDText.com, Inc. Halwani, Z., Jiang, X.-F., Verstraelen, H. and Swidsinski, S. (2014). Infection Reid, G. (2018). Is bacterial vaginosis a disease? Appl. Microbiol. Biotechnol. 102, through structured polymicrobial Gardnerella biofilms (StPM-GB). Histol. 553-558. Histopathol. 29, 567-587. Rodriguez-Garcia, M., Biswas, N., Patel, M. V., Barr, F. D., Crist, S. G., Tchou, I., Misery, L., Sabido, O., Dezutter-Dambuyant, C., Bourlet, T., Moja, P., Ochsenbauer, C., Fahey, J. V. and Wira, C. R. (2013). Estradiol reduces Hamzeh, H., Peguet-Navarro, J., Schmitt, D. and Genin, C. (2001). Functional susceptibility of CD4+ T cells and macrophages to HIV-infection. PLoS ONE 8, HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by e62069-e62069. HIV strain X4. J. Leukoc. Biol. 70, 313-321. Rose, W. A., McGowin, C. L., Spagnuolo, R. A., Eaves-Pyles, T. D., Popov, V. L. Tevi-Bénissan, C., Bélec, L., Lévy, M., Schneider-Fauveau, V., Si Mohamed, A., and Pyles, R. B. (2012). Commensal bacteria modulate innate immune Hallouin, M. C., Matta, M. and Grésenguet, G. (1997). In vivo semen-associated responses of vaginal epithelial cell multilayer cultures. PLoS ONE 7, e32728. pH neutralization of cervicovaginal secretions. Clin. Diagn. Lab. Immunol. 4, Ross, J. A. and Agwanda, A. T. (2012). Increased use of injectable contraception in 367-374. sub-Saharan Africa. Afr. J. Reprod. Health 16, 68-80. Trifonova, R. T., Lieberman, J. and van Baarle, D. (2014). Distribution of immune Round, J. L. and Mazmanian, S. K. (2009). The gut microbiota shapes intestinal cells in the human cervix and implications for HIV transmission. Am. J. Reprod. immune responses during health and disease. Nat. Rev. Immunol. 9, 313-323. Immunol. 71, 252-264. Roxby, A. C., Fredricks, D. N., Odem-Davis, K., Ásbjörnsdóttir, K., Masese, L., United Nations Department of Economic and Social Affairs Population Fiedler, T. L., De Rosa, S., Jaoko, W., Kiarie, J. N., Overbaugh, J. et al. (2016). Division. (2015). Trends in contraceptive use worldwide 2015. Changes in vaginal microbiota and immune mediators in HIV-1-seronegative van de Wijgert, J. H. H. M., Verwijs, M. C., Turner, A. N. and Morrison, C. S. kenyan women initiating depot medroxyprogesterone acetate. JAIDS J. Acquir. (2013). Hormonal contraception decreases bacterial vaginosis but oral Immune Defic. Syndr. 71, 359-366. contraception may increase candidiasis. AIDS 27, 2141-2153. Russell, M. W. and Mestecky, J. (2002). Humoral immune responses to microbial Veres, S., Miller, L. and Burington, B. (2004). A comparison between the vaginal

infections in the genital tract. Microbes Infect. 4, 667-677. ring and oral contraceptives. Obstet. Gynecol. 104, 555-563. Disease Models & Mechanisms

14 REVIEW Disease Models & Mechanisms (2018) 11, dmm035147. doi:10.1242/dmm.035147

Verstraelen, H., Vilchez-Vargas, R., Desimpel, F., Jauregui, R., Vankeirsbilck, in the cervix and vagina of premenopausal and postmenopausal women. N., Weyers, S., Verhelst, R., De Sutter, P., Pieper, D. H. and Van De Wiele, T. Am. J. Reprod. Immunol. 37, 30-38. (2016). Characterisation of the human in non-pregnant Wieser, F., Hosmann, J., Tschugguel, W., Czerwenka, K., Sedivy, R. and Huber, women through deep sequencing of the V1-2 region of the 16S rRNA gene. PeerJ J. C. (2001). Progesterone increases the number of Langerhans cells in human 4, e1602. vaginal epithelium. Fertil. Steril. 75, 1234-1235. Vitali, D., Wessels, J. M. and Kaushic, C. (2017). Role of sex hormones and the Wira, C. R. and Fahey, J. V. (2008). A new strategy to understand how HIV infects vaginal microbiome in susceptibility and mucosal immunity to HIV-1 in the female women: identification of a window of vulnerability during the menstrual cycle. AIDS genital tract. AIDS Res. Ther. 14, 39-39. 22, 1909-1917. Vodstrcil, L. A., Hocking, J. S., Law, M., Walker, S., Tabrizi, S. N., Fairley, C. K. Wira, C. R., Fahey, J. V., Sentman, C. L., Pioli, P. A. and Shen, L. (2005). Innate and Bradshaw, C. S. (2013). Hormonal contraception is associated with a and adaptive immunity in female genital tract: cellular responses and interactions. reduced risk of bacterial vaginosis: a systematic review and meta-analysis. PLoS Immunol. Rev. 206, 306-335. ONE 8, e73055. Wira, C. R., Ghosh, M., Smith, J. M., Shen, L., Connor, R. I., Sundstrom, P., Vodstrcil, L. A., Twin, J., Garland, S. M., Fairley, C. K., Hocking, J. S., Law, M. G., Frechette, G. M., Hill, E. T. and Fahey, J. V. (2011). Epithelial cell secretions from Plummer, E. L., Fethers, K. A., Chow, E. P. F., Tabrizi, S. N. et al. (2017). The the human female reproductive tract inhibit sexually transmitted pathogens and influence of sexual activity on the vaginal microbiota and Candida albicans but not Lactobacillus. Mucosal Immunol. 4, 335-342. Wira, C. R., Rodriguez-Garcia, M., Shen, Z., Patel, M. and Fahey, J. V. (2014). The clade diversity in young women. PLoS ONE 12, e0171856. role of sex hormones and the tissue environment in immune protection against Wagner, R. D. and Johnson, S. J. (2012). Probiotic lactobacillus and estrogen HIV in the female reproductive tract. Am. J. Reprod. Immunol. 72, 171-181. effects on vaginal epithelial gene expression responses to Candida albicans. Wira, C. R., Rodriguez-Garcia, M. and Patel, M. V. (2015). The role of sex J. Biomed. Sci. 19, 58-58. hormones in immune protection of the female reproductive tract. Nat. Rev. Walther-António, M. R. S., Chen, J., Multinu, F., Hokenstad, A., Distad, T. J., Immunol. 15, 217-230. Cheek, E. H., Keeney, G. L., Creedon, D. J., Nelson, H., Mariani, A. et al. Woods, M. W., Zahoor, M. A., Dizzell, S., Verschoor, C. P. and Kaushic, C. (2016). Potential contribution of the uterine microbiome in the development of (2018). Medroxyprogesterone acetate-treated human, primary endometrial endometrial cancer. Genome Med. 8, 122. epithelial cells reveal unique gene expression signature linked to innate Wee, B. A., Thomas, M., Sweeney, E. L., Frentiu, F. D., Samios, M., Ravel, J., immunity and HIV-1 susceptibility. Am. J. Reprod. Immunol. 79, e12781-e12781. Gajer, P., Myers, G., Timms, P., Allan, J. A. et al. (2018). A retrospective pilot Xiu, F., Anipindi, V. C., Nguyen, P. V., Boudreau, J., Liang, H., Wan, Y., Snider, study to determine whether the reproductive tract microbiota differs between D. P. and Kaushic, C. (2016). High Physiological concentrations of progesterone women with a history of infertility and fertile women. Aust. New Zealand reverse estradiol-mediated changes in differentiation and functions of bone J. Obstetrics Gynaecol. 58, 341-348. marrow derived dendritic cells. PLoS ONE 11, e0153304. Wessels, J. M., Lajoie, J., Vitali, D., Omollo, K., Kimani, J., Oyugi, J., Cheruiyot, Zhou, X., Brown, C. J., Abdo, Z., Davis, C. C., Hansmann, M. A., Joyce, P., J., Kimani, M., Mungai, J. N., Akolo, M. et al. (2017). Association of high-risk Foster, J. A. and Forney, L. J. (2007). Differences in the composition of vaginal sexual behaviour with diversity of the vaginal microbiota and abundance of microbial communities found in healthy Caucasian and black women. ISME J. 1, Lactobacillus. PLoS ONE 12, e0187612. 121-133. White, H. D., Crassi, K. M., Givan, A. L., Stern, J. E., Gonzalez, J. L., Memoli, Zhou, J. Z., Way, S. S. and Chen, K. (2018). Immunology of the uterine and vaginal V. A., Green, W. R. and Wira, C. R. (1997a). CD3+ CD8+ CTL activity within the mucosae. Trends Immunol. 39, 302-314. human female reproductive tract: influence of stage of the menstrual cycle and Zhu, J., Hladik, F., Woodward, A., Klock, A., Peng, T., Johnston, C., Remington, menopause. J. Immunol. 158, 3017-3027. M., Magaret, A., Koelle, D. M., Wald, A. et al. (2009). Persistence of HIV-1 White, H. D., Yeaman, G. R., Givan, A. L. and Wira, C. R. (1997b). Mucosal receptor-positive cells after HSV-2 reactivation is a potential mechanism for immunity in the human female reproductive tract: cytotoxic T lymphocyte function increased HIV-1 acquisition. Nat. Med. 15, 886-892. Disease Models & Mechanisms

15