REVIEW ARTICLE: HIV Infection in the Female Genital Tract: Discrete Influence of the Local Mucosal Microenvironment
Charu Kaushic, Center for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L7P4M9.
Citation Kaushic C, Ferreira VH, Kafka JK, Nazli A. HIV infection in the female genital tract: discrete influence of the local mucosal microenvironment. Am J Reprod Immunol 2010
Women acquire HIV infections predominantly at the genital mucosa through heterosexual transmission. Therefore, the immune milieu at female genital surfaces is a critical determinant of HIV susceptibility. In this review, we recapitulate the evidence suggesting that several distinctive innate immune mechanisms in the female genital tract (FGT) serve to significantly deter or facilitate HIV-1 infection. Epithelial cells lining the FGT play a key role in forming a primary barrier to HIV entry. These cells express Toll-like receptors and other receptors that recognize and respond directly to pathogens, including HIV-1. In addition, innate biological factors produced by epithelial and other cells in the FGT have anti-HIV activity. Female sex hormones, co-infection with other pathogens and components in semen may also exacerbate or down-modulate HIV transmission. A combination of innate and adaptive immune factors and their interactions with the local microenvironment determine the outcome of HIV transmission. Improving our understanding of the female genital microenvironment will be useful in developing treatments that augment and sustain protective immune responses in the genital mucosa, such as microbicides and vaccines, and will provide greater insight into viral pathogenesis in the FGT.
According to recent estimates, women constitute >50% of the 40 million people currently living with HIV worldwide.1 In fact, the fastest growing phase of the pandemic is heterosexual transmission in women. Although vaginal intercourse carries a lower HIV transmission probability per exposure event, it contributes more new HIV cases than anal intercourse or parenteral inoculation.2 Recent estimates suggest that 30–40% of annual worldwide HIV infections occur through heterosexual transmission via the female genital tract (FGT).1,2 Given these statistics, it is becoming increasingly clear that a better understanding of HIV interactions in the FGT is critical to developing strategies for prevention of heterosexual HIV transmission. This review will focus on selected aspects that could influence the outcome of heterosexual exposure to HIV-1 and are unique to the local mucosal microenvironment of the FGT.
Innate Barriers in the Female Genital Tract to HIV-1 Infection
For HIV-1 to establish a productive infection in the FGT, it must first evade a number of intrinsic mechanical, chemical and biological barriers. The structure of the FGT forms the first line of defense against HIV. The FGT can be divided into two major compartments: the lower reproductive tract, consisting of the vagina and ectocervix, lined by stratified squamous epithelium; and the upper reproductive tract consisting of the endocervix, endometrium and fallopian tubes, lined by a single layer of columnar epithelium.3 The tight junctions between the columnar cells of the endocervix and endometrium form a mechanical barrier, preventing pathogens from breaching the epithelium. In the vagina and ectocervix, the continuous sloughing of the superficial layers of the stratified epithelium prevents many pathogens from colonizing and establishing infections, providing a better mechanical protection against HIV invasion than the single layer columnar epithelium that lines the upper reproductive tract. However, the greater surface area of the vaginal wall and ectocervix arguably allows greater access for HIV entry, particularly when breaches occur in the epithelium, such as during sexual intercourse.2,4,5
Epithelial cells (ECs) of the FGT produce several biological factors that create an inhospitable environment for HIV including a hydrophilic surface layer of glycoproteins and glycolipids called the glycocalyx, and thick hydrophobic glycoprotein mucus.6 Both the glycocalyx and the mucus act as mucosal barriers and may play a variety of important physiological functions. For example, human cervicovaginal mucus obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1000-fold more slowly than it would in water.7 Several innate immune proteins secreted from ECs with anti-HIV activity are also present within the secretions of the FGT. Those with established anti-HIV properties include secretory leukocyte protease inhibitor (SLPI), lactoferrin, beta (β)-defensins and trappin-2/elafin. The antileukoprotease SLPI is secreted by resident ECs and infiltrating leukocytes in the FGT.8,9 It has been suggested to play an important role in genital mucosal defenses against HIV because of its potent ability to inhibit HIV infection in vitro.10–12 Similarly, lactoferrin, a protein found in breast milk and the genital tract, has also been shown to inhibit HIV at the early stages of viral infection in vitro, by blocking viral adsorption and uptake.13,14 Defensins, a family of small cationic proteins produced in the genital tract, have demonstrated significant antimicrobial effects in studies.15,16 Alpha (α)-defensins are produced by neutrophils, macrophages and γδ T cells, whereas β-defensins are mainly produced by ECs, such as those of the genital mucosa.17 Human β-defensin-1 (HBD-1) is expressed constitutively while HBD-2 and HBD-3 are inducible and have been shown to inhibit HIV-1, particularly X4 tropic strains.18–20 More recently, the serine protease inhibitor, trappin-2/elafin has also been implicated in anti-HIV activity at mucosal surfaces.21,22
In addition to antimicrobial peptides, cells of the FGT can produce interferons (IFNs), which have a wide variety of antiproliferative, immunomodulatory and antiviral effects. Type I interferons (IFN-α, IFN-β) impede the HIV replication cycle through numerous mechanisms, including induction of the antiviral molecule apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G).23,24 Type I IFNs also induce the production of inactive 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which activates a latent endoribonuclease, RNase L, that degrades viral and cellular RNAs resulting in inhibition of protein synthesis.25,26 The 2′,5′-OAS/RNase L pathway has been shown to inhibit the replication of HIV-1.27,28 Protein kinase R, inducible nitric oxide (NO) synthase, myxovirus (Mx)-family proteins and 9–27 proteins are other interferon-inducible proteins shown to have anti-HIV properties.29 A number of studies have shown the inhibitory effect of type II IFN (IFN-γ) on HIV-1 replication in monocytes, monocyte-derived macrophage and lymphocytes.30–32 However, some studies have found a stimulatory effect of IFN-γ on HIV-1 infection.33,34 The recently described type III IFN (IFN-λ, or IL-28/29), which has similar antiviral properties to type I IFN, has been shown to block HIV-1 infection of macrophages by the upregulation of CCR5 ligands (MIP-1α, MIP-1β), as well as anti-HIV proteins like APOBEC3G/3F and type I IFNs.35
The expression of Toll-like receptors (TLRs) by cells of the FGT bestows on them the ability to innately sense their environment for pathogenic motifs and rapidly relay messages to other innate and adaptive cells should a pathogenic breach occur. Vaginal and cervical EC lines express TLRs 1–3, 5 and 6, while primary endocervical ECs express TLRs 1–3 and 6.3 Primary human uterine ECs express TLRs 1–9, indicating the potential of upper reproductive tract to respond to a wide range of pathogens. TLR-mediated activation leads to production of chemokines and cytokines, including IL-6, IL-8, SDF-1 by ECs, as well as resident immune cells in the FGT, allowing for immediate responsiveness to pathogens.36–39 Similarly, the β-chemokines MIP-1α, MIP-1β and RANTES are all secreted by ECs of the upper and lower genital tract constitutively and following infection.40–42 As natural ligands for the CCR5 receptor, these may play a role in blocking R5-tropic HIV-1.
The induction of antiviral responses by activation of TLR pathways in genital ECs also provides a unique potential for utilizing TLR ligands as innate microbicides. To test this possibility, we recently examined the antiviral responses induced in genital ECs following treatment with TLR ligands. The ability of eight different TLR ligands to induce antiviral responses in genital ECs against herpes simplex virus type 2 (HSV-2) infection was determined.43 TLR3 [poly (I:C)], TLR9 (CpG A) and TLR5 ligands (flagellin) showed the greatest ability to reduce HSV-2 replication. Poly (I:C) treatment not only induced maximum interferon-β and NO, but also enhanced production of inflammatory cytokines IL-1α, IL-6 and TNF-α. Similar effects on the inhibition of human cytomegalovirus (CMV) replication in human genital tissues were also recently reported.44 These studies clearly demonstrate that the FGT is equipped with a number of innate defenses against reproductive tract pathogens, including HIV.
HIV interactions with epithelial barrier and target cells in the genital tract
If the intrinsic barriers of the FGT, described earlier, are overcome, HIV-1 is capable of traversing the genital epithelium and establishing an infection. HIV virions have been suggested to cross the epithelium through several pathways, including direct infection of ECs,45 transcytosis of viral particles across the epithelium46–48 and penetration of the virus through epithelial breaches.4,5 HIV-1 can infect both ECs from the lower49,50 and upper FGT.45,48 The nature of viral entry into ECs are likely distinct from the canonical HIV-1 entry pathways as genital ECs demonstrate inconsistent or no expression of CD4 and the chemokine co-receptors CCR5 and CXCR4.49,51,52 In lieu of these molecules, ECs may facilitate HIV transmission using cell surface glycosphingolipids, sulphated lactosylceramide expressed by vaginal ECs53 and galactosylceramide expressed by ectocervical ECs,52 which have been found to bind HIV-1 gp120 and foster transcytosis. Interactions of HIV-1 gp120 with transmembrane heparin sulfate molecules, such as syndecans, expressed by genital ECs may also contribute to HIV-1 attachment and entry.47,50 A variant of salivary agglutinin named gp340, which is expressed on cervical and vaginal ECs, has also been implicated in the passage of HIV through the epithelium.54,55 The relative contribution of these receptors to HIV entry and infection in genital ECs is unclear.
In addition to ECs, there are a number of resident immune cells in the FGT that may also contribute to HIV transmission, most notably dendritic cells (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.56 However, the relative contribution of different types of DCs is not completely understood. Langerhans cells in the cervicovaginal epithelium express CD4 and CCR5, but not CXCR4 or the surface adhesion molecule DC-SIGN, which has been shown to assist in HIV transmission at mucosal surfaces. In a study using ex vivo human organ culture system, HIV-1 rapidly infected both intraepithelial vaginal Langerhans and CD4+ T cells. HIV-1 entered CD4+ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, resulting in productive infection. By contrast, HIV-1 entered CD1a+ Langerhans cells primarily by endocytosis, and virions persisted intact within these cells for several days without active replication.57 In contrast, a recent study was unable to detect translocation of HIV-1 in reconstructed human vaginal mucosa, and presence of Langerhans cells did not alter HIV-1 transmission.58 These studies suggest that the main target cells for HIV-1 are CD4+ DCs and T cells in the lamina propria of genital mucosa. Consequently, the enriched population of CD4+ T cells and APCs present in the transformation zone, where the ectocervix transitions into the endocervix, may be a particularly susceptible site for HIV entry.59
In addition to the interactions described earlier, we recently reported a novel mechanism that may allow HIV to breach the epithelial barrier of the intestinal and genital mucosae, resulting in translocation of both HIV-1 and other luminal microbes.60 In this study, we demonstrated that HIV-1 surface glycoprotein could directly reduce transepithelial resistance, a measure of epithelial monolayer integrity, by 30–60% in primary genital epithelial and intestinal cell lines cultures. The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers and was mediated by direct response of ECs to the envelope glycoprotein of HIV-1 seen by upregulation of inflammatory cytokines that lead to impairment of barrier functions. The increased permeability could be responsible for small but significant migration across the mucosal epithelium by virus and bacteria present in the lumen. This mechanism could be particularly relevant to mucosal transmission of HIV-1.
Hormonal contraception and altered susceptibility to HIV infection
The regulatory effects exerted by the cyclic presence of sex hormones confer the female reproductive tract with a unique microenvironment. Estradiol and progesterone play a key role in regulating physiology and functions of the FGT, including immune responses (reviewed in3). Therefore, the use of hormonal contraception needs to be carefully studied, as long-term, or even short-term administration of these hormones may have far-reaching effects on host responses. Long-acting, progesterone-based contraceptives, such as depot medroxy-progesterone acetate, are highly effective and currently used by more than 100 million women worldwide.61 Multiple studies in humans and rhesus macaques suggest that the use of progesterone-based formulations may predispose one to increased risk of HIV-1 or SIV infection, higher viral burden and increased viral shedding.62–64 A recent study further suggested that women who use progesterone-based contraceptives display accelerated HIV-1 disease progression and mortality, compared to women who do not.65 Although the pathways involved in these outcomes are not clear, progesterone is known to regulate a number of immunological pathways, including the inhibition of CTLs and natural killer cells.66–68 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.69,70 In contrast to progesterone, estrogen and its derivatives may exert a strong protective effect against HIV-1 infection.71 Systemic administration of estrogen in the form of subcutaneous implants protected against intravaginal challenge of ovariectomized female rhesus macaques with highly pathogenic SIVMAC251.72 While these studies suggest that hormone-based contraceptives may perturb the mucosal environment resulting in altered HIV-1 susceptibility, the mechanisms underlying these observations need to be clearly elucidated. This is because several studies have failed to observe an overall effect of hormonal contraception on the incidence of HIV-1 infection,73,74 while other studies report increased risk of infection, but only in subgroups of subjects differing in age and HSV-2 status.75,76 Interpretation of these studies is complicated by multiple factors (type, dose and method of administration of hormonal contraceptives) and design of studies (cross sectional, longitudinal).
To gain a better understanding of mechanism by which hormones regulate susceptibility in the FGT, we have conducted studies in a model of genital herpes infection. Using this model, we demonstrated that long-acting progestational formulation Depo-Provera increased susceptibility to genital HSV-2 infection by 100-fold.77 Further studies indicated that longer progesterone treatment regimes resulted in poor mucosal immune responses and increased susceptibility.78 In other studies, mice were ovariectomized and treated with exogenous estradiol and progesterone prior to primary infection with genital herpes or immunization with attenuated strain of herpes virus. The results from these studies indicate that estradiol treatment regulates susceptibility while progesterone treatment leads to increased chronic inflammation and pathology.79,80 Further support for the role of estradiol in controlling pathology comes from a more recent study where mice immunized with attenuated HSV-2 via subcutaneous or intranasal route under the influence of estradiol, developed remarkably decreased pathology compared to progesterone-treated mice, following genital challenge.81 These findings were recently confirmed using an HSV-2 vaccine formulation.82
Co-infections in the female genital tract
Sexually transmitted infections (STI) and other genital infections have been associated with increased HIV genital shedding, transmission and susceptibility.83,84 These may include CMV, gonorrhea, syphilis, bacterial vaginosis, candidiasis and genital herpes. Bacterial STIs, such as Chlamydia and gonorrhea have been epidemiologically associated with increased subsequent HIV acquisition and, by extension, with increased sexual transmission of HIV.85,86 The increased HIV susceptibility may relate to local micro-ulcerations because of the pathologies associated with the infection or to the local recruitment of activated immune cells, which may act as targets for HIV.87 HSV-2 is one of the most prevalent STIs, infecting 20–30% of sexually active adults in North America and over 70% in sub-Saharan Africa.88 A recent meta-analysis demonstrated HSV-2 infection to be associated with a threefold increase in susceptibility to HIV by both men and women from the general population.89 Part of this increased susceptibility is more likely attributed to HSV-2-induced ulcerations, which creates a breach in the physical barrier of the genital epithelium.90 Genital HIV-1 shedding is also markedly increased during clinical HSV-2 reactivations, accompanied by an increase in HIV-1 plasma viral load.91 Other studies have shown that, among individuals shedding both viruses, the amounts of viral shedding are closely related.92 Herpetic lesions and possibly asymptomatic HSV-2 mucosal shedding generates an influx of activated CD4+ T cells that persist for months after healing, which may facilitate the transmission of HIV.93 HSV-2 replication is also associated with a 10-fold increase in the number of immature DCs expressing DC-SIGN and a threefold increase in CCR5 expression on CD4+ T cells.94 Therefore, it is possible that HSV-2 infection may increase the number of HIV-target cells in the FGT, facilitating the transmission of HIV. Certain immediate early proteins of HSV-1 such as infected cell protein (ICP)-0 and ICP4 have been shown to interact with the HIV-1 LTR to induce HIV replication,95 suggesting that HSV can directly upregulate HIV replication. Recent observations in our laboratory show that genital ECs infected with HSV-2 or treated with an array of TLR ligands, representative of various bacterial and viral pathogens, are capable of inducing HIV replication, directly and indirectly (V.H. Ferreira and C. Kaushic, unpublished). Altogether, these studies demonstrate that co-infections may directly or indirectly enhance HIV acquisition and transmission.
Influence of semen on HIV transmission
Semen represents the main vector of HIV dissemination, as transmission occurs more efficiently from men to women, and men to men than from women to men.96 It is composed of cells and secretions from the testes, epididymis, prostate, seminal vesicles and bulbourethral gland, and it has been reported to enhance HIV infection.97 A number of components of seminal fluid, whose physiological purpose is to protect spermatozoa, also protect HIV virions. For example, basic amines such as spermine, spermidine, putrescine and cadaverine commonly protect both spermatozoa and HIV virions from the threat of acid inactivation in the vaginal tract.97 In recent studies, fractionation of semen from healthy donors has led to the identification of a semen-derived enhancer of viral infection (SEVI). SEVI consists of amyloid fibrils composed of internal 34–40 amino acid proteolytic fragments from prostatic acid phosphatase, a protein present at a concentration of approximately 1–2 mg/mL in semen that can enhance HIV infection up to 105-fold in cell culture.97,98
Semen also plays an active role in transforming the molecular and cellular environment of the FGT. Studies suggest that vaginal epithelium secretes the chemokine CCL20 in response to seminal plasma, which enhances recruitment of Langerhans cells to the vaginal mucosa. This may facilitate the transport of virions across the vaginal epithelium barrier to the lymph nodes.99 It was also found that the HT-29 human EC line was sensitive to HIV-1 in the presence of whole semen resulting in a two-fold increase in infectivity.100 Others have shown that seminal plasma can upregulate expression of proinflammatory cytokines in human genital epithelium raising the possibility of the role of seminal plasma in enhancing STI, including HIV-1, in the FGT.101,102 Transforming growth factor β1 (TGF-β1) concentration in human seminal plasma is one of the highest measured in biological fluids.103 When deposited into the FGT, TGF-β in seminal plasma plays both proinflammatory and immunosuppressive roles in preparing the FGT for the conceptus.103 We have recently observed that semen from men infected with HIV may contain different concentrations of TGF-β, depending on the stage of infection, and this in turn may induce differential responses in the FGT (J.K. Kafka and C. Kaushic, unpublished).
The FGT is a unique mucosal environment and a key target site for heterosexual HIV-1 transmission. The mucosal ECs are key sentinels that serve the dual function of forming the primary barrier as well as being the first responders to HIV-1 in the event of a breach. Recent studies show that these cells express TLRs and other receptors that can respond directly and rapidly to HIV-1 exposure. Several other biological factors secreted in the FGT, such as SLPI, lactoferrin, defensins and trappin/elafin, provide intrinsic protection against HIV-1 infection. Discrete factors such as female sex hormones, other co-infections and semen may be present differentially in the microenvironment of the FGT and play a key role in determining the susceptibility of the host. The combination of these and other protective mechanisms and factors that confer susceptibility in the FGT likely determine the net outcome of HIV-1 exposure. Understanding the FGT microenvironment and its interactions with HIV-1 can assist in the development of better strategies to enhance innate and adaptive immunity and develop novel methods to prevent HIV-1 infection.