Influence of Common Mucosal Co-Factors on HIV Infection in the Female Genital Tract

Introduction

The global demographics of HIV and AIDS have changed dramatically over the course of the past 30 years. It was first discovered as a disease that primarily affected men who have sex with men (MSM) and intravenous drug users but has evolved into an epidemic where currently approximately half of 34 million adults living with HIV globally are women.1 Although vaginal intercourse carries a lower HIV transmission probability per exposure event than anal intercourse or parenteral inoculation,2 the female genital tract (FGT) has been estimated to be the predominant site of HIV acquisition globally, with approximately 40% of all new infections originating at this mucosal site.2, 3 Thus, it has become increasingly clear that a better understanding of the microenvironment in the FGT is critical for developing strategies for the prevention of HIV transmission globally.

Transmission of HIV in the female genital tract

The FGT can be divided into two major compartments: the lower genital tract (LGT), consisting of the vagina and ectocervix, lined by stratified squamous epithelium; and the upper genital tract (UGT) consisting of the endocervix, endometrium, and fallopian tubes, lined by a single layer of columnar epithelium.4 Tight junction proteins between the columnar epithelium of the UGT form a mechanical barrier, preventing pathogens from breaching the protective lining. In the stratified epithelium of the LGT, continuous sloughing of dead superficial genital epithelial cells (GECs) prevents many pathogens from colonizing or establishing infections. The multilayered squamous epithelium, when intact, may likely provide better mechanical protection against HIV invasion than the single-layer columnar epithelium that lines the UGT. However, the greater surface area of the vaginal wall and ectocervix arguably allows more access sites for HIV entry,2 particularly when breaches occur in the epithelium, such as during sexual intercourse.5, 6

Despite much debate, there is no clear consensus regarding the primary site of transmission of HIV-1 in the FGT. Although tiny microabrasions in the LGT are a common occurrence during normal sexual intercourse and could provide an easy portal for viral transmission,7 other studies, particularly those that examined acute simian immunodeficiency virus (SIV) infection in non-human primates8, indicate that HIV may preferentially invade through the UGT and local viral amplification likely precedes systemic dissemination. Adding further credence to this concept is the fact that a large number of activated CD4⁺ T-cells populate the cervical transformation zone, providing a rich source of target cells for HIV-1.9 Furthermore, a recent study using human tissue found that HIV-1 could penetrate both intact human cervical columnar and squamous epithelial barriers to depths where the virus could encounter potential target cells.10

Natural barriers to HIV infection in the female genital tract

The FGT contains a number of endogenous barriers that provide protection against HIV acquisition. GECs of the FGT produce several biological and chemical factors that create an inhospitable environment for HIV including a hydrophilic surface layer of glycoproteins and glycolipids called the glycocalyx, and a thick hydrophobic glycoprotein mucus.11 Both the glycocalyx and mucus act as mucosal barriers against HIV-1 and other pathogenic microbes. A recent study demonstrated that human cervicovaginal mucus obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse 1000 times more slowly than it would in water.12 GECs, as well as resident immune cells, such as macrophage and dendritic cells (DCs), also secrete innate antimicrobial peptides (AMPs) with anti-HIV activity. These include secretory leukocyte protease inhibitor (SLPI), α- and β-defensins as well as trappin-2/elafin.13 More recently, anti-proteases, such as serpins and cystatins expressed by GECs, have also been shown to inhibit HIV binding and replication and reduce local inflammation.14

In addition to AMPs, cells of the FGT can produce interferons (IFNs) that have a wide variety of immunomodulatory and antiviral effects. Type I IFNs (IFN-α, IFN-β) impede HIV replication by several mechanisms, including inducing the upregulation of restriction factors such as apolipoprotein B mRNA-editing enzyme–catalytic polypeptide-like 3G (APOBEC3G),15, 16 tripartite motif 5α (TRIM5α),17 bone marrow stromal antigen 2 (BST2; also known as tetherin)18 SAM domain and HD domain 1 (SAMHD1)19, 20, and myxovirus resistance 2 (MX2 also known as MxB).21 Interestingly, type I IFN has also been implicated as a contributor to HIV pathogenesis22; increased type I IFN is a component of the signature associated with chronic immune activation.23 The benefit/harm of IFN responses may likely depend on the net outcome of a number of factors, including the stage of infection. Evidence from our laboratory suggests that in response to HIV-1 gp120, GECs significantly upregulated IFN-β and neutralization of this IFN-β resulted in enhanced induction of the HIV long terminal repeat (LTR) promoter in transfected Jurkat T-cells (A. Nazli, V.H. Ferreira & C. Kaushic, unpublished results). These results suggest that early type I IFN responses originating at the site of transmission may play a role in reducing HIV replication in the FGT, in contrast to the effects of IFN that take place during the chronic stages of infection which may contribute to immune activation.

Two new mucosal IFN species have recently been described to possess anti-HIV activity. Unlike other type I IFNs, IFN-ε is expressed constitutively in mucosal tissues including the reproductive tract.24 IFN-ε is the only type I IFN family member to be expressed by HeLa cells.25 Moreover, seminal plasma was also found to upregulate expression of IFN-ε in cervicovaginal tissues,26 suggesting that IFN-ε may play a protective role in reproductive tissue. Interestingly, when IFN-ε was used in an intranasal/intramuscular heterologous HIV prime-boost immunization, increased HIV-specific CD8 T-cell responses were observed in the spleen, genito-rectal draining lymph nodes, and Peyer's patches.27 Furthermore, the recently described type III IFN-λ (IL-28/29), which has similar antiviral properties to Type I IFN, has been shown to block HIV-1 infection in macrophages in vitro28, 29 by inhibiting HIV-1 integration and post-transcriptional events.30 Interestingly, IFN-λ receptors are largely restricted to cells of epithelial origin. Together these results suggest that IFN-ε and IFN-λ may play a unique role in protecting the genital mucosa and future explorations of their potential role in protecting the FGT against HIV may prove valuable in the context of vaccine or microbicide development.

In addition to the innate factors described above, resident immune cells as well as non-immune cells of the FGT, such as GECs, express various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which allows them to sense foreign microbes in their environment and rapidly relay messages to other innate and adaptive immune cells. Primary endocervical GECs express TLRs 1–3 and 6.4 In addition, primary human uterine GECs express TLRs 1–9, indicating the potential to respond to a wide range of pathogens. Expression of NOD1 and NOD2 has also been detected in the human endometrium.31 PRR recognition of pathogens typically initiates an intracellular signaling cascade resulting in the activation of transcription factors such as NFκB and the production of a variety of cytokines and chemokines.32 Many of these cytokines and chemokines also have the ability to directly interfere with HIV infection. The chemokine stromal-derived factor-1 (SDF-1 or CXCL12), which is found within the subepithelial layer of the cervix, is able to inhibit X4 strains of HIV competitively.33 Similarly, the β-chemokines macrophage inflammatory protein 1-α (MIP-1α), MIP-1β, and regulated on activation, normal, T-cell expressed, and secreted (RANTES) are all secreted by cells of the upper and lower genital tracts constitutively and under infectious conditions,34-36 and, as natural ligands for the CCR5 receptor, may also play a role in blocking R5-tropic viruses from establishing an infection.

Epithelial barrier in FGT during HIV infection

The mucosal barrier formed by the epithelial cells of the FGT forms the first line of defense against the entry of pathogens. Sexually transmitted organisms, including HIV-1, have to breach this barrier to establish infection. There is evidence that during HIV infection, both the intestinal and genital mucosal barriers are disrupted and memory CD4⁺ T-cells at these mucosal surfaces are severely depleted.37-39 HIV-1 infection is characterized by chronic systemic inflammation, seen by increased levels of serum lipopolysaccharide (LPS) and soluble CD14 (sCD14) in HIV-1-infected individuals.37, 40-44 HIV-1 disease progression has been correlated with increased circulating levels of LPS, an indicator of microbial translocation, which is associated with mucosal barrier disruption.43 The chronic immune activation associated with HIV disease is considered to be one of the main driving forces leading to immunodeficiency. The etiology of microbial translocation associated with HIV infection is not clearly understood and has been linked to impairment in the mucosal epithelial barrier. Our recent work supports this premise by demonstrating that primary human GECs directly interact with HIV-1 surface glycoprotein gp120 leading to production of an array of proinflammatory cytokines.45 Among these cytokines, TNF-α production induced a rapid decrease in transepithelial resistance (TER), a measure of epithelial barrier integrity. The disruption in the barrier was accompanied with increased mucosal permeability as well as bacterial and viral translocation across the epithelium. Thus, increased mucosal permeability and microbial translocation could result directly from early interactions between HIV-1 and the genital epithelium leading to initiation of microbial translocation and initiating immune activation. Further studies have since revealed that gp120-mediated activation of proinflammatory cytokine pathways in GECs utilizes TLR2 and TLR4 in addition to cell surface heparan sulfate moieties.46 Further ongoing studies are examining the in vivo relevance of gp120-mediated increased permeability.

More recently, a role for local immune factors such as IL-22 in the maintenance of barrier function in the intestinal mucosa in both human and SIV models has emerged. IL-22 is a member of the IL-10 family of cytokines with epithelial reparative and regenerative properties.47 Recent studies suggest that the absence of IL-22 contributes to HIV-1 and SIV pathogenesis.48, 49 Kim et al48 recently demonstrated that during chronic HIV-1 infection, IL-22-producing Th22 cells were severely depleted, and this was accompanied by compromised epithelial integrity in the intestinal mucosa. Further, in vitro IL-22 treatment protected intestinal epithelial cells against HIV-1- or TNF-α-induced barrier dysfunction. Similar IL-22-mediated protection is also observed in GECs (A. Nazli & C. Kaushic, unpublished results). These studies indicate that local immune factors in the mucosa could play an important regulatory role in epithelial barrier maintenance related to HIV-1 pathogenesis.

HIV interactions with cells of the female genital tract

If the intrinsic barriers in the FGT, described above, are overcome, HIV-1 is capable of crossing the genital epithelium and establishing an infection. HIV virions have been suggested to traverse the epithelium through several pathways, including direct infection of GECs50; transcytosis of viral particles across the epithelium51-53; and penetration of the virus through epithelial breaches.5, 6 While there is evidence that HIV-1 can infect GECs from the LGT54, 55 or UGT,50, 53 these findings are contentious, particularly as to whether these epithelial cells are productively infected. In agreement with some of the earlier work done on epithelial cell lines, a recent study reported that ectocervical and endocervical epithelial cell lines became productively infected with cell-free HIV-1 in a CD4-independent manner and that this infection increased when inoculation occurred in the presence of semen-derived enhancer of virus infection (SEVI) fibrils.56 Consequently, the de novo virus was transmitted to target CD4 T-cells in co-culture in a contact-dependent manner. In recent studies performed in our laboratory, exposure of primary human endometrial and endocervical GECs to R5 or X4 tropic strains of HIV-1 did not result in the detection of HIV-1 pro-viral DNA integration, RNA splicing or reverse transcription products, although virus was seen to be taken up by endocytosis into GECs, suggesting that primary human GEC could be non-productively infected (V.H. Ferreira & C. Kaushic, unpublished results).

The nature of viral entry into GECs is likely distinct from the canonical HIV-1 entry pathways as GECs demonstrate inconsistent expression of CD4 and the chemokine co-receptors CCR5 and CXCR4.54, 57, 58 In lieu of these molecules, GECs may facilitate HIV transmission using cell surface glycosphingolipids, sulfated lactosylceramide expressed by vaginal GECs59, and galactosylceramide expressed by ectocervical GECs,58 which have been found to bind HIV-1 gp120 and foster transcytosis. Interactions of HIV-1 gp120 with transmembrane heparan sulfate proteoglycans, such as syndecans, expressed by GECs, may also contribute to HIV-1 attachment and entry.52, 55 A variant of salivary agglutinin named gp340, which is expressed on cervical and vaginal GECs, has also been implicated in the passage of HIV through the epithelium.60, 61 The relative contribution of these receptors to HIV entry and infection in GECs is unclear.

In addition to GECs, there are a number of resident immune cells in the FGT that may contribute to HIV acquisition or transmission, most notably DCs and T-cells. DCs appear to play a major role in HIV transmission and dissemination, as well as driving the early inflammatory response to infection.62 However, the relative contribution of different types of DCs is not completely understood. Some studies purport that resident Langergans cells (LCs) and CD4⁺ T-cells are the primary target of HIV-1 in the genital tract,3 whereas others have shown that the presence of vaginal LCs does not alter HIV transmission.63 Furthermore, the transformation zone where the ectocervix changes into the endocervix has an enriched population of CD4⁺ T-cells and APCs and may therefore be a particularly susceptible site for HIV acquisition.9

Female sex hormones, hormonal contraception, and altered susceptibility to HIV infection

A number of studies in the last two decades indicate that endogenous female sex hormones and exogenous hormonal contraceptives affect HIV-1 infection or disease progression (reviewed in64). Non-human primate studies have consistently found that the administration of depot medroxyprogesterone acetate (DMPA) to rhesus macaques enhances the risk of SIV acquisition,65-67 whereas estradiol (E2) and its derivatives have been shown to be protective against SIV infection.68 DMPA is a progesterone (P4)-based synthetic contraceptive, used by more than 100 million women around the world, and it is particularly popular in resource-poor settings in the developing world, where HIV rates remain high.69 Human epidemiological studies have shown that DMPA use may lead to increased risk of HIV-1 infection, disease progression, and mortality compared to women who did not use the injectable contraceptive formulation DMPA.65, 70-72 More recently, a prospective cohort study of nearly 3800 serodiscordant couples from seven African nations found that the risk of acquiring HIV from an infected male partner was twice as high among women who used injectable hormonal contraceptives and that HIV-infected women who used injectable hormonal contraceptive were twice as likely to transmit HIV to an uninfected male partner.73 Subsequently, UNAIDS had urgent consultations with an expert panel, and although guidelines for hormonal contraceptive were not subsequently altered, there was a renewed call for more research on hormonal contraceptives and HIV infection risk.74, 75

Although the pathways involved in these outcomes are not clear, P4 is known to regulate a number of immunological pathways,76 including the inhibition of CTLs77 and natural killer cells.78 It also decreases the production and alters glycosylation of IgG and IgA antibodies, modulates cytokine production, and upregulates HIV-1 receptor expression on CD4⁺ T-cells.79, 80 With respect to DMPA, a recent study found that DMPA use inhibited the production of IFN-γ, IL-2, IL-4, IL-6, IL-12, TNF-α, MIP-1α as well as IFN-α in peripheral blood cells and activated T-cells following stimulation with TLR ligands.81 Others have found that in vaginal biopsies from women receiving DMPA, the numbers of HIV target cells were significantly increased compared to vaginal tissues taken during the follicular and/or luteal phases of untreated cycles.82 In a recent study, cervical tissue explants from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo and among the eight tissues that were productively infected all were obtained from women in their secretory phase (high P4) of their menstrual cycle.83 Together these results suggest that P4 and P4-based contraceptives, in particular DMPA, may play a significant role in regulating susceptibility to genital tract infections, such as HIV-1, and underscore a need to elucidate the underlying mechanisms involved in this regulation.

To gain a better understanding of the mechanism by which hormones regulate susceptibility in the FGT, we have extensively studied a mouse model of genital herpes infection. Using this mouse model, we demonstrated that DMPA increased susceptibility to genital herpes simplex virus type 2 (HSV-2) infection by 100-fold.84 Further studies indicated that prolonged DMPA treatment regimes resulted in poor mucosal immune responses and increased susceptibility.85 In other studies, mice were ovariectomized and treated with exogenous E2 and P4 prior to primary infection with genital herpes or immunization with attenuated strain of herpes virus. The results from these studies indicate that E2 treatment regulates susceptibility while progesterone treatment leads to increased chronic inflammation and pathology.86, 87 Subsequent studies demonstrated the protective effect of E2 in intranasal immunized mice that had significantly decreased pathology compared to P4-treated mice.88 These findings were confirmed by other studies, using an HSV-2 vaccine formulation.89

Recent studies in our laboratory have examined whether endogenous female sex hormones or hormonal contraceptives regulate GEC susceptibility to HIV-1. Our results indicate that HIV-1 is internalized within GECs via endocytosis. However, no early or late reverse transcription products, integrated HIV-1 pro-viral DNA, or spliced HIV RNA transcripts were measured, regardless of the presence or absence of hormones. The results suggest that female sex hormones, particularly DMPA, may regulate HIV entry and transcytosis, but not replication in GECs (V.H. Ferreira & C. Kaushic, unpublished results). Ongoing studies are investigating the significance of DMPA-enhanced HIV entry and transcytosis in the absence of productive infection.

Co-infections in the female genital tract

STIs have been associated with increased HIV genital shedding, transmission, and susceptibility.90, 91 Such infections may include gonorrhea, syphilis, bacterial vaginosis, candidiasis, and genital herpes. Bacterial STIs, such as Chlamydia trachomatis and Neisseria gonorrhea, have been epidemiologically associated with increased subsequent HIV acquisition, and, by extension, with increased sexual transmission of HIV.92, 93 The increased HIV acquisition may relate to local micro-ulcerations due to the pathologies associated with the infection, or to the local recruitment of activated immune cells, which may act as targets for HIV.94 Bacterial vaginosis (BV) is a common disorder characterized by changes in vaginal flora in which normally predominant Lactobacillus species are replaced by potential pathogens including Gardnerella vaginalis, genital Mycoplasma, and fastidious anaerobic bacteria.95, 96 BV has been associated with a 60% increased risk of HIV-1 acquisition in women,97 and, among women with HIV-1, with higher HIV-1 concentrations in cervicovaginal fluids.98 Bacteria associated with BV can induce viral replication and shedding in the genital tract,99 which may lead to increased HIV-1 infectiousness for women with BV.100

Herpes simplex virus type 2 is one of the most prevalent STIs, infecting 20–30% of sexually active adults in North America and up to 80% of adults in sub-Saharan Africa.101 A previous meta-analysis indicated HSV-2 infection to be associated with a threefold increase in susceptibility to HIV by both men and women from the general population.102 Part of this increased susceptibility is likely due to HSV-2-induced ulcerations, which create a breach in the physical barrier of the genital epithelium.103 Genital HIV-1 shedding is also markedly increased during clinical HSV-2 reactivations, accompanied by an increase in HIV-1 plasma viral load.104 Herpetic lesions and possibly asymptomatic HSV-2 mucosal shedding generate an influx of activated CD4⁺ T-cells that persist for months after healing, which may facilitate the transmission of HIV.105

In response to sexually transmitted co-infections, cells in the genital tract, including GECs, upregulate inflammatory responses. Proinflammatory cytokines have been implicated in enhancing HIV infection at the cellular level. Studies of latently infected target cells have shown that the addition of cytokines such as TNF-α, IL-6, or IL-1β increases HIV replication, mediated through the HIV-LTR.106-108 Previously, we showed that in response to common co-infecting STIs, specifically HSV-1, HSV-2, and N. gonorrhea, primary GECs upregulated proinflammatory cytokines including TNF-α, IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1), which contributed to indirect induction of the HIV-LTR promoter in T-cells, a process synonymous with HIV replication. Furthermore, by blocking inflammatory signaling pathways, either with the broad anti-inflammatory compound curcumin or with specific NFκB and AP-1 inhibitors, this indirect induction could be blocked.109 Inflammation may therefore play a major role in the acquisition or spread of HIV-1 infection.110 Interestingly, it has been previously observed that lower levels of IL-1β, IL-6, and TNF-α were measured in unstimulated PBMCs of highly exposed persistently seronegative (HESN) women, suggesting an immunoquiescent phenotype among this resistant cohort.111 In another study, HIV genital tract shedding was significantly associated with higher cervico-vaginal lavage (CVL) concentrations of IL-6, IL-1β, MIP-1α, and RANTES.112 Thus, a future exploration of using anti-inflammatory compounds for the purpose of protecting the epithelial barrier-disrupting function of HIV-1 gp120 as well as the inflammation associated with co-infecting microbes may therefore present a novel and valuable new modality for preventing HIV infection in the FGT or curbing the spread of the infection.

Influence of semen on HIV transmission

Semen is the main vector of HIV dissemination as transmission in FGT occurs primarily following exposure to virus-containing seminal fluid during sexual intercourse.113-116 Semen contains a plethora of factors, which serve to enhance or inhibit HIV infection. For example, spermatozoa can capture virus through heparan sulfate and transmit it to DCs, thus increasing their infection.117 In contrast, clusterin and mucin-6 in seminal plasma (SP) can compete with HIV as DC-SIGN ligands.118-120 Several in vitro studies reported the existence of amyloid fibrils, formed by amyloidogenic fragments of prostatic acid phosphatase and semenogelins found abundantly in semen, which may increase the infectivity of HIV by several orders of magnitude by facilitating virion attachment to target cells.121-125 In vivo, SP and cationic SEVI may facilitate the spread of physiologically lower doses of HIV-1 found during sexual transmission,126; however, further in vivo studies are needed to test this hypothesis. In contrast, other cationic polypeptides such as semenogelin contribute to anti-HIV activity in SP.127 Furthermore, deposition of semen into the acidic vaginal environment can raise the pH, which may decrease116, 128 or increase129 the infectivity of the virus.

The influence of semen on the FGT microenvironment can have a profound effect on HIV transmission. When semen from HIV-uninfected men is deposited into the FGT, it elicits a transient inflammatory response characterized by proinflammatory cytokine and chemokine production and immune cell recruitment, which serves to prime the FGT for the conceptus.26, 130-133 However, this response may also create an environment favorable for HIV infection as SP has also been shown to induce the overexpression of COX-2 in mare and porcine endometrium134, 135 and enhance COX-2 expression in human vaginal ECs,136 thus promoting an inflammatory environment. Additionally, vaginal GECs have been shown to upregulate the chemokine CCL20 in response to SP, which promoted the migration of LCs into the vaginal mucosa.137 Interestingly, when it comes to transmigration of cell-associated virus across FGT epithelium, SP decreased epithelial crossing of immune cells by increasing their adherence to the epithelial monolayer, and increasing TER.138

The presence of HIV infection in the male genital tract (MGT) can alter semen composition, which can have a profound effect on FGT responses to semen. This includes changing the cytokine network and further enriching SP with cytokines which can modulate HIV replication139 and promote HIV shedding and local target cell activation.140 The immunoregulatory factor transforming growth factor beta-1 (TGF-β1) is found in very high concentrations in human SP and functions to induce FGT tolerance to the allogeneic fetus.130 Because of its abundance in semen and its immunoregulatory role, we examined the role of seminal TGF-β on FGT responses and how these responses may differ during acute and chronic stages of HIV infection. Our results showed that seminal plasma from HIV-uninfected and HIV-infected antiretroviral therapy (ART)-naive men in acute stages of infection contained higher levels of proinflammatory cytokines and lower levels of TGF-β1 compared to ART-naive men in chronic stages of infection, which leads to an increase in proinflammatory cytokine production from endometrial GECs.141 GEC responses to SP regardless of the presence or stage of HIV infection increased HIV-LTR expression, suggesting that SP can promote HIV replication in infected target cells in the FGT.141 More recently, we found that TGF-β1 was compartmentalized between blood and semen, and latent TGF-β1 was positively correlated with the immune activation marker sCD14 in SP, suggesting that activated monocytes and macrophages in the MGT may co-express TGF-β1 and sCD14 in response to HIV infection. Interestingly, sCD14 was negatively correlated with high levels of active TGF-β1 seen in SP of chronic ART-naive men. This suggests that high levels of active TGF-β1 could play a role in regulating chronic immune activation, thus furthering our understanding of the role of TGF-β1 in HIV transmission (J.K. Kafka & C. Kaushic, unpublished results).

Conclusion

The FGT is a key target for HIV-1 transmission in women, and the outcome of exposure to HIV is likely determined by a number of co-factors that influence this mucosal microenvironment (Fig. 1). The combination of innate physical barriers such as tight junctions and mucus in the mucosal epithelial lining as well as chemical barriers including the anti-microbial peptides and HIV-1 restriction factors can have a direct anti-viral effect. They can also indirectly decrease HIV-1 infection and replication by inducing an anti-inflammatory microenvironment that is unfavorable for HIV. Other co-factors that may be present in the FGT, such as viral and bacterial co-infections, bacterial vaginosis, and direct interactions with HIV, disrupt the mucosal barrier, can directly and indirectly facilitate HIV-1 infection and replication. They can also create a microenvironment favorable for HIV infection and replication by attracting target T-cells into the FGT, increasing immune activation, barrier disruption, and microbial translocation. Other factors in the FGT milieu including semen, Type I IFN, and sex hormones have been shown to have both types of effects, and more work needs to be done to determine which effects are predominant in vivo. Further complexity is likely conferred in the genital tract because of the interactions between the co-factors present simultaneously in the FGT. Further in vivo studies examining the co-factors will provide insights that can assist in the development of future prophylactic strategies against HIV.