HIV-1 Transmission in the Male Genital Tract

Authors

  • Yonatan Ganor,

    1. Mucosal Entry of HIV-1 and Mucosal Immunity, Cell Biology and Host Pathogen Interactions Department, Cochin Institute, Université Paris Descartes, CNRS (UMR 8104), Paris, France
    2. INSERM, U1016, Paris, France
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  • Morgane Bomsel

    1. Mucosal Entry of HIV-1 and Mucosal Immunity, Cell Biology and Host Pathogen Interactions Department, Cochin Institute, Université Paris Descartes, CNRS (UMR 8104), Paris, France
    2. INSERM, U1016, Paris, France
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Morgane Bomsel, Cochin Institute, INSERM U1016, CNRS UMR8104, Paris Descartes University, 22 rue Me´chain, 75014 Paris France.
E-mail: morgane.bomsel@inserm.fr

Abstract

Citation Ganor Y, Bomsel M. HIV-1 transmission in the male genital tract. Am J Reprod Immunol 2011; 65: 284–291

HIV-1 is mainly a sexually transmitted infection, and epithelial surfaces covering genital mucosa are the primary site of HIV-1 transmission. Although male circumcision was reported to reduce male acquisition of HIV-1 by 60%, the initial mechanisms of HIV-1 transmission in the male genitals remain elusive. We established two novel models of the adult human foreskin epithelium that allowed for polarized infection via the mucosal pole with either HIV-1-infected cells that are present in all secretions vectorizing HIV-1 or cell-free HIV-1. Efficient HIV-1 transmission occurs following 1 hr of polarized exposure of the inner, but not outer, foreskin to mononuclear cells highly infected with HIV-1, but not to cell-free virus. HIV-1-infected cells form viral synapses with apical foreskin keratinocytes, leading to polarized budding of HIV-1, which is rapidly internalized by Langerhans cells (LCs) in the inner foreskin. In turn, LCs form conjugates with T-cells, thereby transferring HIV-1. Seminal plasma from HIV-negative men mixed with cervico-vaginal secretions from HIV-positive women, which mimics the in-vivo mixture of these genital fluids during woman-to-man HIV-1 sexual transmission, decreases HIV-1 infection at the foreskin. Our results rationalize at the cellular level the apparent protective outcome of circumcision against HIV-1 acquisition by men.

Introduction

The Structure and the Role of Mucosal Epithelia in the Initial Steps of HIV-1 Infection

HIV-1 is a viral menace that gains access into the body mainly during sexual intercourse, by crossing epithelial barriers that cover the mucosal surfaces of the gastrointestinal, female, and male genital tracts. These mucosal epithelia have evolved to provide different levels of protection from incoming foreign molecules and pathogens. Stratified epithelia that underlay areas of opening and contact with the external environment are made of multiple layers of epithelial cells,1 providing a high degree of mechanical barrier and defense against bacterial and viral insults originating from the external environment. Moreover, various immune cells, such as Langerhans cells (LCs), T-cells, dendritic cells (DCs), and macrophages (which are either inserted between the epithelial cells or reside within the lamina propria), further enable the specific recognition of pathogens and the subsequent engagement of the immune system to eliminate rapidly and efficiently these invading threats. In contrast, simple epithelia covering mucosal surfaces within the body consist of monolayers of polarized epithelial cells connected by tight junctions,1 as well as various immune cells residing below the epithelial barrier. These simple mono-layered epithelia provide a lower degree of protection.

How does HIV-1 cross epithelial barriers and gain access into the body? Many studies addressing this question have clearly demonstrated that HIV-1 utilizes different mechanisms to enter the body via the mucosa, depending on the type of epithelial barrier it encounters. Thus, it seems that HIV-1 has evolved to ‘highjack’ existing physiological processes in mucosal epithelia to invade the body: pathogen recognition by LCs in stratified epithelia and transcytosis in simple epithelia.

Hence, in the vagina and exocervix stratified epithelia, early HIV-1 transmission involves capture of HIV-1 by epidermal LCs. These professional antigen-presenting cells, integrated within these epithelia, normally sample the mucosal surface for incoming foreign pathogens and then migrate to secondary lymphoid organs to present processed antigens to T-cells.2 LCs are among the first cells to capture HIV-1, because of their close proximity to the mucosal surface and their ability to bind the HIV-1 envelope glycoprotein subunit gp120 via their unique C-type lectin langerin.3–5 At low viral concentrations, this process leads to HIV-1 degradation by LCs.5 However, at higher concentrations of the virus, the protective effect of langerin is inhibited,5 leading to the subsequent transfer of the internalized virus to T-cells and the productive infection of T-cells,6,7 either locally within the epithelium or in the draining lymph nodes following LCs migration.

In contrast, in the rectum, intestine, and endocervix simple epithelia, we and others have shown that HIV-1 crosses in-vitro the epithelial barrier by the transcellular pathway of transcytosis.8–12 This process involves the formation of an apical transient contact, the ‘viral synapse’, between HIV-1-infected cells and epithelial cells, which induces polarized virus budding at the contact area between the two cell types.8,11,13,14 The newly budded virus is then rapidly internalized by the epithelial cells (without productively infecting them, as is the case in-vivo), transcytosed towards the basal pole, and released still infectious to the basal environment.8

Transmission of Cell-associated versus Cell-free HIV-1

Early studies including our own have shown that HIV-1 transcytosis across simple epithelia is much more efficient, when HIV-1 particles bud locally at viral synapses compared to cell-free HIV-1.8,11,13–16 Notably, all genital fluids that transmit HIV-1, such as seminal plasma (SP) and cervicovaginal secretions (CVS), contain HIV-1-infected cells. For example, SP contains on the order of 105 white blood cells/mL with substantial numbers of macrophages and CD4+ T-cells.17 In HIV-1-infected men, the infection rate of this seminal HIV-1-susceptible cell population was estimated at 0.2%.17 Similarly, several studies documented HIV-1-susceptible host cells in CVS and demonstrated the presence of HIV-1 DNA or RNA and the ability to culture the virus from the cell-associated fractions.17 A recent study showed that systemic and persistent infection could be achieved in female macaques following vaginal exposure to SIV-infected cells.18 These observations clearly emphasize the importance of cell-associated HIV-1 as a major and efficient transmission vector. Yet, most research on mucosal HIV-1 transmission has focused on cell-free HIV-1, probably because of the ease in preparing and manipulating cell-free HIV-1 compared to cell-associated HIV-1. Therefore, further studies are still urgently needed to better characterize the cellular and molecular events underlying cell-associated HIV-1 transmission.

The Biology of Sexual Intercourses and its Relevance to HIV-1 Transmission

As mentioned earlier, sexual transmission of HIV-1 is mediated in-vivo by genital fluids (e.g. SP and CVS). Previous studies have shown that SP induces secretion of chemokines by epithelial cells of the female genital mucosa, leading to subsequent transient attraction of mononucleated cells,19 which may in turn increase the susceptibility to HIV-1 infection. However, recent in-vitro studies investigating the effect of SP on HIV-1 transmission have reached contradictory conclusions, demonstrating that endogenous factors in SP may either enhance20 or inhibit21 HIV-1 infection and transfer to T-cells. Other studies have shown that CVS contain a plethora of protective innate factors against HIV-1.22–30 Although these studies clearly suggest that genital fluids may directly affect HIV-1 transmission, their role in mucosal HIV-1 infection models is rarely evaluated.

Importantly, the sexual transmission of HIV-1 is also facilitated by several sexually transmitted diseases (STDs). Longitudinal epidemiological studies have provided direct evidence that STDs in HIV-1-uninfected individuals increase their risk of acquiring HIV-1.31,32 However, it has been difficult to determine which STD has the greatest effect on HIV-1 transmission. For instance, Chlamydia trachomatis and Neisseria gonorrhoeae are common exclusive human STD pathogens, which primarily infect the urogenital epithelia, i.e. the urethra in men and the cervix in women, and cause mucosal inflammation.33–36 Both pathogens can invade their target epithelial cells, resulting in production of proinflammatory cytokines by the infected cells and subsequent influx of various immune cells to the infection site.35,37,38 While this process may further increase the amount of potential HIV-1 target cells, the exact details of the interplay between various STD pathogens and HIV-1 in mucosal epithelia are to a large extent unknown.

The foreskin epithelium and HIV-1

Male Circumcision Protects Against HIV-1

Three recent reports from randomized clinical trials of male circumcision in Africa concluded that circumcision offers a 60% reduction in HIV-1 transmission from infected women to men.39–41 These studies provided the first direct evidence of the efficacy of male circumcision in protecting men against HIV-1 infection. This protective effect was consistent with both ecological and epidemiologic studies, which also showed a protective effect of 50–70% in men at high risk of HIV-1 infection.42 Such observations suggest that the foreskin epithelium in uncircumcised men may be highly permissive to HIV-1 and may serve as an efficient entry portal for the virus.

The Structure of the Foreskin Epithelium

The adult human foreskin is a stratified epithelium made of keratin-producing epithelial cells. The foreskin consists of two different aspects, outer and inner, which are easily distinguished by the relative decrease in melanocytes in the inner foreskin.43 Early studies have reported that the outer foreskin is highly keratinized, while the inner foreskin shows a much lower degree of keratinization.44,45 However, later studies reached other results: a study of foreskin keratinization in Chinese boys and adults reported that the extent of keratinization was much greater in the inner foreskin;46 another study reported no difference between the keratinization of the inner and outer foreskins.47 In our own experiments, we repeatedly observed at the ultra-structural level a higher number of apical keratin layers in the outer foreskin, which were thicker and usually denser compared to those in inner foreskin (Fig. 1). The discrepancies mentioned earlier may be attributed to the lack of a standardized method to evaluate keratin thickness and further suggest that age and genetic factors may affect the degree of foreskin keratinization.

Figure 1.

 Keratinization of the foreskin epithelium. Electron microscopy images of the apical surface of inner (a) and outer (b) foreskins. The consecutive numbers represent the different layers of terminally differentiated foreskin keratinocytes that form the apical deposit of keratin. Of note, many keratin layers in outer foreskin are thicker compared to inner foreskin. Scale bars = 2 μm.

Limited Information Exists Regarding HIV-1 Transmission in the Male Genitals

Importantly, potential HIV-1 target cells and receptors are present in the foreskin (Fig. 2). Cells expressing CD4 (i.e. LCs and T-cells), the principal receptor for HIV-1, and the co-receptor CCR5 are present in the inner and outer foreskin. Other possible HIV-1 target cells are also present within the dermis below the epithelial barrier (i.e. macrophages and DCs).43–45,48–51

Figure 2.

 HIV-1 target cells in the inner foreskin. (a) Sections of inner foreskin were stained with a goat-anti-human antibody to langerin, followed by a secondary FITC-conjugated anti-goat IgG antibody (green). Cell nuclei were counterstained with DAPI (blue). The white dotted line shows the basement membrane; scale bar = 10 μm. The single starred frame is shown below in higher magnification and demonstrates that the LC dendrites reach the apical surface. (b) Electron microscopy image of a LC within the epidermis; scale bar = 2 μm. The left frame (marked 1) is shown in higher magnification below and to the left and demonstrates the presence of a typical Birbeck granules (BG) with a racket shape (black arrowhead); scale bar = 200 nm. The right frame (marked 2) is shown in higher magnification below and to the right and demonstrates the presence of typical BGs with a rod shape (black arrows); scale bar = 100 nm. (c) Electron microscopy image of a T-cell within the epidermis, positioned above the basement membrane (black dotted line); scale bar = 1 μm. (d) Electron microscopy image of a macrophage within the dermis, surrounded by collagen fibers; scale bar = 1 μm.

Relevant to HIV-1, the higher extent of outer foreskin keratinization has been suggested to provide a protective barrier against HIV-1 transmission.44,52 Surprisingly, only two previous studies have investigated the mechanism of foreskin HIV-1 entry. In these two studies, only the entry of high doses of cell-free HIV-1 at time points of >24 hr was evaluated. In the first report, upon infection of agarose-sealed foreskin tissue explants, the inner foreskin appears susceptible to infection with R5, but not X4, cell-free HIV-1.44 However, sealing efficiency and thereby polarization of the infection in this system have been previously questioned.50 In the second report, using non-polarized foreskin tissue explants where HIV-1 can gain access to both apical and basal tissue surfaces, inner and outer foreskins were infected to a similar degree with R5, but not X4, cell-free HIV-1.50 While these observations suggest that the stratified foreskin epithelium is indeed susceptible to HIV-1, a precise description of the initial events of foreskin HIV-1 transmission remains elusive.

Mechanisms of HIV-1 entry and transmission in the foreskin epithelium

Development of Novel Models of the Stratified Foreskin Epithelium

A major problem in studying HIV-1 transmission in humans is the lack of proper in-vitro viral transmission model systems that reflect the complex in-vivo structure of such epithelia. This is even further complicated by the fact that non-primate animals are resistant to HIV-1, and experiments with non-human primates are expensive and time elaborating. Thus, as a crucial step towards better understanding of the initial events of mucosal HIV-1 invasion, one has to develop appropriate models of these epithelia. A relevant model should mimic as closely as possible the structural and morphological features of normal epithelia and should also contain all the cell types that consist the epithelium, namely epithelial cells and various immune cells.

Indeed, previous studies have aimed at modeling various human mucosal epithelia, and two methodological approaches have been routinely used: ex-vivo tissue explants and in-vitro reconstruction. In the first approach, biopsies originating from various mucosal tissues are mounted in two-chamber systems that provide restricted access to either apical/mucosal or basal compartments.6,9,44,53–58 However, such explant models are not optimal: the function and integrity of the mucosal barrier are maintained only for few hours; the surgical procedure itself allowing for tissue sampling activates the migratory immune cells in these epithelia, leading to their rapid exit out of the explants; efficient sealing of the explant tissue edges to ensure polarization of the infection (i.e. from the apical to the basal compartment, as takes place in-vivo) is not always met. In the second approach, we and others have previously developed in-vitro models of tight polarized simple epithelial barriers, by cultivating monolayers of epithelial cells onto permeable supports in two-chamber systems.8,13,59–61 However, such models differ remarkably from the in-situ situation, as neither the stratified architecture is reconstituted nor the presence of immune cells.

To describe the initial events of HIV-1 entry at the foreskin, we recently developed two novel and complementary models of the adult human foreskin epithelium.62 First, in an improved foreskin explant model, pieces of normal adult human foreskin tissues from either inner or outer foreskin are placed with their epidermal/apical side facing up on top of a permeable membrane in a two-chamber system. To restrict HIV-1 exposure via the apical side, hollow plastic cloning ring cylinders are adhered tightly to the epidermal surface of each tissue explant, using surgical glue. This approach results in the creation of a highly sealed apical chamber that permits for subsequent polarized inoculation of HIV-1. Second, an alternative 3D immuno-competent in-vitro model of the foreskin epithelium was established, based on our previous successful reconstruction of an immuno-competent vaginal epithelium63 and on methodologies used thus far for the development of skin equivalents.64 Briefly, primary inner or outer foreskin fibroblasts and keratinocytes are sequentially seeded in the apical compartment of a two-chamber system, together with immature LCs/DCs. Culture medium and conditions are optimized to allow distinct patterns of differentiation of the foreskin keratinocytes, to mimic the structure of either the low keratinized inner or highly keratinized outer foreskin epidermis. This experimental approach results in the establishment of novel immuno-competent in-vitro models of both inner and outer foreskins, with the structural and morphological characteristics of normal foreskin.

Early Steps of HIV-1 Foreskin Entry

By using the newly developed foreskin models and various morphological and quantitative experimental approaches, we recently described the ‘chain-of-events’ mediating early HIV-1 entry in the foreskin during the first hour of viral exposure:62

(i) Efficient HIV-1 transmission occurs following 1 hr of polarized exposure of the inner, but not outer, foreskin epithelium. The higher degree of keratinization in the outer foreskin provides a physical/mechanical barrier against HIV-1 entry, while the lower degree of keratinization in the inner foreskin facilitates the entry of HIV-1 particles into the epidermis. Hence, fluorescent and confocal microscopy studies of foreskin explants demonstrate that HIV-1 particles are trapped within the thick apical keratin layer of the outer foreskin, but are able to penetrate into the epidermis of the inner foreskin. HIV-1 translocation studies in foreskin reconstructions show that virus translocates to a much lower degree across outer compared to inner foreskin.

(ii) Efficient foreskin HIV-1 transmission occurs when the virus originates from HIV-1-infected cells compared to cell-free virus. The infectious potential of cell-associated versus cell-free HIV-1 has been little evaluated in stratified epithelia.53,55,63 In the newly developed foreskin models, HIV-1-infected cells form viral synapses with apical foreskin keratinocytes, leading to polarized budding of HIV-1 at the contact area between the two cells types, as evident by ultra-structural microscopy studies of foreskin explants and reconstructions. Subsequently, potent HIV-1 translocation occurs when induced by HIV-1-infected cells, while cell-free HIV-1 translocates poorly, as evident by HIV-1 entry studies in foreskin reconstructions.

(iii) LCs and T-cells are the initial targets of HIV-1 during foreskin transmission. During 1hr exposure to HIV-1, LCs modify their spatial distribution within the foreskin epidermis, sample HIV-1, and transfer the virus to T-cells. Thus, in inner foreskin explants, fluorescence and ultra-structural microscopy studies illustrate that epidermal LCs migrate towards the apical surface, rapidly internalize HIV-1 and then transfer the virus to epidermal T-cells across LC-T-cell conjugates (Fig. 3). Flow cytometry studies show increase in LC-T-cell conjugates following HIV-1 inoculation.

Figure 3.

 HIV-1 transfer across cell conjugates. Electron microscopy image of a cell–cell conjugate within the epidermis, positioned above the basement membrane (black dotted line); scale bar = 1 μm. This contact may either represent a LC T-cell conjugate in which the LC transfers HIV-1 to the T-cell, or a conjugate between T-cells that permits the local expansion of an HIV-1-infected founder population. The contact area between the two cells is show in higher magnification and demonstrates the presence of HIV-1 particles (white arrowheads); scale bar = 100 nm.

(iv) A mixture of SP and CVS inhibits HIV-1 foreskin translocation. To evaluate the impact of genital fluids on HIV-1 foreskin entry, the apical poles of inner foreskin reconstructions were inoculated with HIV-1-infected cells (i.e. conditions in which efficient HIV-1 translocation takes part) alone or together with various genital fluids. To mimic the in-vivo events during heterosexual intercourse between uninfected men and HIV-1-infected women, SP from HIV-1-negative men was mixed with CVS from HIV-1-positive women. The presence of either SP or CVS alone has no effect on HIV-1 translocation. Unexpectedly, a mixture of SP and CVS decreases foreskin HIV-1 entry.

Conclusions

Based on our studies, as well as that by others, we propose that the main pathway for foreskin HIV-1 entry results from interaction of HIV-1-infected cells with the inner part of the foreskin. Such contact would lead to viral synapse formation, resulting in HIV-1 budding and capture by epidermal LCs. These cells thus play an active role in sampling HIV-1 at the foreskin followed by transfer of the virus to T-cells. Such infected T-cells might initiate the local expansion of a small founder population of HIV-1-infected cells within the foreskin mucosa, which is a prerequisite for HIV-1 dissemination and systemic infection.65,66 This rapid process may be blocked by yet ill-defined components activated when mixing genital fluids. Moreover, such process is probably driven by molecular signals, such as chemokines, which could be induced by HIV-1 within the foreskin tissue and that may mediate changes in LCs and T-cells spatial distribution in the epidermal and/or dermal compartments. (Zhou et al. 2010, unpublished data).

Together, these findings rationalize at the cellular level the apparent protective outcome of circumcision against HIV-1 acquisition by men: both surfaces of the foreskin are invaded by HIV-1, especially upon contact with HIV-1-infected cells. Thus, removal of the foreskin, especially the inner aspect, following circumcision results in the elimination of a mucosal surface rich in HIV-1 target cells, which serves as an efficient HIV-1 entry portal in men.

Ancillary