Interactions between HIV-1 and Mucosal Cells in the Female Reproductive Tract

Introduction

Human immunodeficiency virus type 1 (HIV-1) enters the host in virtually all transmissions, excluding those acquired parenterally, through the mucosal surfaces of either the genital tract or the gastrointestinal tract.1-3 However, despite remarkable scientific progress during the past three decades, an effective HIV-1 vaccine and strategies to prevent HIV-1 transmission remain elusive, underscoring the need for increased understanding of HIV-1 mucosal transmission. Indeed, many critical issues have not been fully elucidated, including the identification of the initial HIV-1 target cells, parameters of local viral propagation, pathways of viral dissemination, and the mechanism of R5 selection.

Worldwide, the heterosexual route is the prevalent mode of HIV-1 transmission,1, 2 with the risk of transmission 0.03–0.5% per coital act.4-6 Pertinent to this review, the female reproductive tract accounts for approximately 40% of all HIV-1 transmissions.3 As in other routes of mucosal infection, HIV-1 infection in the female reproductive tract involves three major events: entry through the mucosal epithelium, productive infection in subepithelial mononuclear cells, and delivery to lymph nodes to initiate systemic infection. Recent studies, as previously reviewed,3, 7-21 have expanded but not completely defined the immediate events that follow HIV-1 exposure in the genital mucosa. Here, we provide a focused review of the interactions between HIV-1 and key mucosal cells, including epithelial cells, lymphocytes, macrophages, and dendritic cells (DCs), in the female reproductive tract in heterosexual transmission. A summary of these interactions is listed in Table 1. In addition to our discussion of these events, we emphasize several unresolved issues that warrant future investigation, including the role of macrophages in HIV-1 dissemination and in latency.

HIV-1 entry through genital mucosal epithelium

The first event in mucosal HIV-1 infection is translocation of the virus across the epithelium. Among persons at risk for HIV-1 infection, disruption of cervicovaginal mucosa induced by trauma or infection-associated mucosal inflammation, ulceration, and erosions provides HIV-1 access to lymphoid cells in the subepithelial lamina propria (Figs 1 and 2), resulting in higher rates of HIV-1 transmission.22 In the absence of mucosal disruption due to trauma or infection, possible cellular routes of HIV-1 entry into the mononuclear cell-rich lamina propria include epithelial cells, lymphocytes, Langerhans' cells, myeloid DCs, and macrophages (Figs 1 and 2).23-25

Human immunodeficiency virus-1 entry in the female reproductive tract may occur via vagina, ectocervix, endocervix, uterus, and possibly Fallopian tubes, regions with distinct epithelial architecture (Table 1). The epithelium of the endocervix, uterus, and Fallopian tubes, like that of the gut, is a single layer of polarized, columnar epithelial cells with tight junctions, dividing the epithelium into apical and basolateral domains (Fig. 2).2, 26 In contrast, the epithelium of the vagina and ectocervix is composed of multilayered, pluristratified epithelial cells that do not have a polarized plasma membrane or tight junctions (Fig. 1).2, 6, 26, 27 These distinct features may promote different translocation processes in different regions of the female genital tract (Figs 1 and 2). In the vaginal and ectocervical mucosae, the absence of epithelial cell tight junctions likely permits CD4⁺ T cell, macrophage, and DC migration into the vaginal and ectocervical epithelium, where the cells may take up HIV-1 that has entered the ‘leaky’ epithelium. In this regard, we have shown that vaginal macrophages25 and myeloid DCs (manuscript submitted) take up HIV-1 in a vaginal mucosa explant model. We also have shown that vaginal DCs transport virus across the vaginal mucosa (manuscript submitted). Moreover, using vaginal and ectocervical mononuclear leukocytes isolated from human vaginal and ectocervical tissues provided by healthy women undergoing reconstructive pelvic surgery, we have demonstrated that vaginal and ectocervical macrophages, myeloid DCs, and lymphocytes take up HIV-1.23 In elegant studies, Hladik, et al.24 induced vaginal blisters to show that intraepithelial CD4⁺ T cells and Langerhans' cells translocate HIV-1 across the vaginal epithelium. As in the intestinal mucosa,26, 28-32 transcytosis and translocation of HIV-1 by epithelial cells or surface-penetrating DCs are the likely cellular routes by which HIV-1 enters the lamina propria of the human endocervical mucosa.

Transcytosis of virions through squamous epithelial cells has been suggested,3 but classic transcytosis occurs in polarized, columnar epithelial cells and not non-polarized, pluristratified squamous epithelium.26 Thus, the recent identification of the neonatal Fc receptor (FcRn) in the basal layer of human vaginal epithelium33, 34 and ectocervical epithelium34 and in the columnar epithelial cells that line the human endocervix35 and uterus,33 as well as the penile urethra,34 underscores the potential role of this receptor in HIV-1 entry in genital mucosa. The FcRn substantially enhances HIV-1 transcytosis across an endometrial epithelial cell monolayer in the presence of both an acidic environment and Env-specific IgG.34 These data indicate that FcRn-mediated transcytosis could contribute significantly to male-to-female and male-to-male transmission, because genital and rectal secretions from infected persons contain Env-specific antibodies.35-37

Productive infection in subepithelial mononuclear cells

After translocation across the mucosal epithelium, HIV-1 encounters potential target cells in the dense lymphocyte and macrophage populations of the lamina propria (Table 1). Lamina propria CD4⁺ T lymphocytes in human and macaque vaginal and cervical mucosae are early target cells for HIV-1 and SIV and support viral replication.23, 24, 38-41 Vaginal, as well as ectocervical, macrophages also have been shown to support HIV-1 replication.23, 25, 42 Macrophages from vaginal and ectocervical mucosa were similar in their capacity to support infection, as were lymphocytes from vaginal and ectocervical mucosa. However, when lymphocytes were compared to macrophages from the same mucosal compartment, lymphocytes supported more robust replication than the macrophages.23 Furthermore, isolated mixed cell cultures and vibratome sections from human uterus, Fallopian tube, ectocervix, and cervix are permissive to HIV-1.43 Although DCs alone do not support HIV-1 replication, DC-T-cell conjugates support high levels of viral replication.

Epithelial cells in HIV-1 mucosal infection of the female reproductive tract

Genital epithelial cells have been shown to support transcytosis of cell-free virus across epithelium in primary human endocervical epithelial cells and genital mucosa-derived cell lines, including VK2/E6E7, Endo1/E6E7, Ect1/E6E7, and HEC1A that are derived from vaginal, endocervical, ectocervical, and endometrial tissue, respectively.44, 45 Nazli et al.46 reported that primary endometrial and cervical epithelial cells interact directly with HIV-1 via gp120, inducing upregulation of an array of pro-inflammatory cytokines. Pro-inflammatory cytokines such as TNF-α in turn lead to the impairment of barrier functions, promoting HIV-1 entry across the epithelium and access to lymphoid cells in lamina propria. Furthermore, human primary uterine epithelial cells,47 uterine epithelial cell lines,48 and ectocervical epithelial cell line Ect1/E6E749 capture and transmit HIV-1 to CD4⁺ T cells. In contrast, Micsenyi et al.50 reported that ectocervical (Ect1/E6E) and endocervical (End1/E6E7) epithelial cell line cells are capable of transmitting HIV-1 to CD4⁺ T cells only when de novo HIV-1 is produced within the epithelial cells.

Genital epithelial cell permissiveness to HIV-1 infection is controversial. Human primary epithelial cells isolated from uterus,43, 47, 51 ectocervix,44 cervix,43, 51 Fallopian tube,52 as well as human uterine (RL95-2, HEC1A, and ECC1),48 ectocervical (Ect1/E6E7) and endocervical (End1/E6E7) epithelial cell line cells,50 support HIV-1 replication. Human cervix-derived epithelial cell line ME180 also can be productively infected by cell-associated HIV-1 and remains infected for 8 months, suggesting that cervical epithelial cells support HIV infection.52-54 In sharp contrast, others have reported that isolated human primary ectocervical and endocervical epithelial cells do not support productive HIV-1 infection by cell-free or cell-associated HIV-1.42, 55 As noted by the authors, the non-permissiveness of cervical primary epithelial cells to HIV-1 in these studies is likely related to the absence of surface expression of HIV-1 receptor and co-receptors.56 The absence of HIV-1 receptor and co-receptors on isolated cervical primary epithelial cells may be due to the enzyme used to isolate the cells. Dispase, for example, has been shown to cleave CD4 from lymphocytes after 1-hr incubation, whereas collagenase has no effect after 3-hr incubation.56

Dendritic cells in HIV-1 mucosal infection of female reproductive tract

Studies of monocyte-derived DCs (MoDCs), blood DCs, Langerhans' cells, and myeloid DCs in the simian immunodeficiency virus (SIV)/rhesus macaque non-human primate model have provided many insights into the critical role of DCs in HIV-1 transmission.7, 57-60 However, DCs in different tissues and mucosal compartments display distinct phenotypes and functionality, precluding the simple extrapolation of findings to DCs in the female reproductive tract. Study of the interactions between HIV-1 and human mucosal DCs in the female reproductive tract has been hindered by the limited availability of human female genital tissue and the difficulty in isolating mucosal cells.

In the macaque model, SIV inoculated into the macaque vagina enters the mucosa within 60 min through intraepithelial DCs and can be detected in draining lymph nodes within 18 hr.61 The presence of DC-SIGN on genital and gut DCs61 may facilitate DC transport of virus in the mucosa. Although Langerhans' cells do not express DC-SIGN or CCR5, they may participate in early HIV-1 uptake, as shown in macaques inoculated intravaginally with SIV.59 The location of Langerhans' cells in the upper layer of the stratified epithelium positions these cells for the uptake of free virions that have penetrated this region of the squamous epithelial barrier.

Mucosal DCs consist of myeloid DCs, plasmacytoid DCs, and Langerhans' cells. In human studies, attention has focused on Langerhans' cells, which have been shown to take up HIV-1 in vaginal epithelial sheets,24, 62 human skin explants,63 and epidermal cells isolated from human skin.64 Recently, we showed that human intestinal lamina propria myeloid DCs rapidly take up HIV-1, transport the virus through the mucosa, and transmit virus in trans to peripheral blood and intestinal lymphocytes.31 In addition, DC-SIGN⁺ cells from human rectal mucosa have been shown to bind and transfer HIV-1 to peripheral blood CD4⁺ T cells.65 In female genital mucosa, myeloid DCs and Langerhans' cells have been shown to take up and/or transfer HIV-1 to CD4⁺ T cells.23, 24 Vaginal mucosa contains more than 10-fold less plasmacytoid DCs, compared to myeloid DCs (manuscript submitted). Hladik and colleagues24 showed that HIV-1 rapidly penetrates intraepithelial CD1a⁺ Langerhans' cells, which reside in the genital tract epithelium. Among vaginal and ectocervical mononuclear cells, DCs are the first cells to take up HIV-1.23 As early as 15 min after virus inoculation, 5.1% of vaginal myeloid DCs and 1.7% of ectocervical myeloid DCs contain virus. In contrast, vaginal and ectocervical macrophages first contain detectable HIV-1 at 2 hr.23 Notably, myeloid DCs in human vaginal lamina propria differ phenotypically from MoDCs, take up HIV-1 up to 10-fold more virus than MoDCs, transport HIV-1 through the mucosa, and then trans-infect vaginal and peripheral blood target cells (manuscript submitted).

Lymphocytes in HIV-1 mucosal infection of female reproductive mucosa

The identification of CD4⁺ T cells in the human vaginal epithelium and documentation of HIV-1 penetration into lymphocytes in the epithelial sheets obtained by suction blister24 suggest that CD4⁺ T cells may be involved in HIV-1 entry into vaginal mucosa. In suspension culture of mucosal mononuclear cells, vaginal and ectocervical lymphocytes take up HIV-1.23 These cells first displayed detectable HIV-1 uptake at 2 hr, much later compared with DC uptake of the virus.23

In human and macaque gastrointestinal mucosa, most attention has focused on the small intestine, where lamina propria CD4⁺ T cells are prominent HIV-1 and SIV target cells and undergo profound depletion shortly after infection.66-72 In human vaginal mucosa, CD3⁺ T cells are scattered throughout the lamina propria and infrequently at the basal region of the squamous epithelium. Lymphocytes also are detected at the edges of dermal papillae.25 HIV-1 has been detected in CD4⁺ T cells in vaginal epithelium in a modified organ culture system by electron microscopy.24 We also have shown that vaginal, as well as ectocervical, lymphocytes support HIV-1 replication.23, 25 Similarly, CD4⁺ T cells in macaque vaginal mucosa support HIV-1 replication and are rapidly depleted following intravenous inoculation of SIV.41 In human and macaque cervical mucosa, lamina propria CD4⁺ T lymphocytes also are early target cells for HIV-1 and SIV and support viral replication.23, 38-40 Howell et al.43 reported that isolated mixed cell cultures and vibratome sections from human uterus, Fallopian tubes, ectocervix, and cervix are permissive to HIV-1, but the cells responsible for HIV-1 replication were not identified.

Macrophages in HIV-1 mucosal infection of female reproductive mucosa

Monocytes and macrophages play a fundamental role in the transmission, establishment, pathogenesis, and persistence of HIV-1 infection.73-75 Evidence that macrophages participate in the transmission of HIV-1 derives from the identification of HIV-1-infected tissue macrophages in the body fluids that transmit HIV-1, including semen, cervical fluid, and colostrum/early breast milk.76-80 Among suspension of mucosal mononuclear cells, vaginal and ectocervical macrophages capture HIV-1 and first display detectable virus uptake at 2 hr.23 In a vaginal explant model, HIV-1 was inoculated onto the apical surface of explanted vaginal mucosa, and 30 min later, the explants were harvested, sectioned, stained, and analyzed by confocal microscopy for macrophages that contain virus. HIV-1 virions were identified in macrophages,25 indicating that HIV-1 translocates across the epithelium in vaginal mucosa and then is taken up by subepithelial macrophages.

In vaginal mucosa, macrophages are detected in the lamina propria, occasionally in the basal region of the squamous epithelium and at the edges of dermal papillae.25 In cervical mucosa, CD14-/68-positive macrophages are restricted to subepithelial mucosa.42 Ectocervical subepithelial macrophages have been shown to be among the first cells to be infected in ectocervical explants.42 In addition, using isolated vaginal and ectocervical mononuclear cells23 or purified vaginal macrophages,25 we have shown that both vaginal and ectocervical macrophages support HIV-1 replication.

Macrophages in local spread of HIV-1

Productively infected tissue macrophages also likely contribute to the local spread of virus through the transfer of HIV-1 to other target cells. In this connection, HIV-1-infected macrophages have been shown to transmit virus to CD4⁺ lymphocytes by fusing with the T cell81 and through the formation of transient viral synapses between viral Gag and Env on the macrophage and CD4 on the T cell.82, 83 Macrophage transfer of HIV-1 to T cells may serve to augment CD4⁺ T-cell infection and subsequent depletion.84 In addition, the macrophage mannose-binding receptor appears to be capable of capturing and transferring virus to T cells through a non-fusigenic mechanism.85 Notably, Vpx-dependent viral amplification in macrophages at sites of virus inoculation appears to be a prerequisite for the efficient dissemination of virus in tissue sites.86 Also, the ability of HIV-1-infected monocytes to cross the blood–brain barrier likely contributes to the delivery of HIV-1 to the brain, leading to microglial infection.87 Similarly, blood monocytes are the exclusive source of intestinal macrophages,88 promoting the delivery of systemically infected monocytes to the intestinal mucosa.

Emerging role for macrophages in HIV-1 persistence

During highly active antiretroviral therapy (HAART), latent HIV-1 infection in memory CD4⁺ T cells is a major obstacle to curative therapy. These latent-infected lymphocytes are considered the primary reservoir for latent/persistent HIV-1 infection.89-94 In addition, HIV-1-infected macrophages have gained appreciation as another important population of latently infected cells.93, 95-98 In patients receiving HAART, HIV-1 proviral DNA has been detected in blood monocytes as well as in activated and resting CD4⁺ T cells, but viral DNA decays more slowly over time in monocytes than in T cells. CD16⁺ monocytes appear to constitute a continuing source of viral persistence during HAART.99 Furthermore, sequence evolution and phylogenetic analysis have shown that monocytes, not lymphocytes, are the source of viral replication during and after prolonged HAART,96 implicating mononuclear phagocytes as an important source of latent virus. Consistent with human studies, macrophages have been identified as the principal reservoir of mononuclear cell in macaques infected with SIV/HIV-1 chimeric virus.100 Therefore, macrophages are well suited to serve as reservoir cells in HIV-1 infection due to their remarkable longevity, resistance to HIV-1 cytopathic effects, and ability to evade host immune responses. In this regard, we have reported that intestinal macrophages, unlike monocyte-derived macrophages, are resistant to HIV-1 infection and can carry integrated but transcriptionally silent HIV-1,101-103 indicating that intestinal macrophages are a potential reservoir for latent HIV-1 during HAART. Whether female genital mucosal macrophages function as a potential reservoir for latent HIV-1 is an important topic for future study.

Concluding remarks

Available evidence reviewed here shows that both lower and upper female reproductive tracts are involved in HIV-1 heterosexual transmission (Table 1). The interactions between HIV-1 and mucosal cells in the female reproductive tract are far from being well defined and further investigation is needed. Important topics that warrant investigation include local HIV-1 propagation, pathways of viral dissemination, the mechanism of selection of transmitted/founder viruses, and the influence of the local mucosal microenvironment on each of these events. Increased understanding of the interactions between HIV-1 and mucosal cells in the female reproductive tract will provide critical new insights for the development of effective vaccine and microbicide strategies for the prevention of HIV-1 transmission.