The genital tract is the main route of natural infection for HIV. Understanding the mechanisms of how HIV is transmitted is crucial for the development of effective strategies designed to prevent HIV infection and pandemic. In the initial phase of sexual transmission, the virus crosses mucosal epithelium and is spread to proximal lymphoid organs where a permanent infection is established (reviewed in ).
Multiple hypotheses have been proposed for HIV transmission: disruption of the integrity of the mucosa [2,3], direct infection of epithelial cells , transcytosis across epithelial cells , or virus uptake or infection of immune cells or all . Several studies [7,8] have demonstrated that virus transcytosis occurs within the first 2 h of HIV infection using monostratified intestinal epithelial cells. By contrast, ex-vivo genital mucosa organ cultures showed that cervix epithelial cells are not susceptible to infection and do not transcytose virions, and that transmission is mediated by immune cells [9,10]. Indeed, in female rhesus monkeys, Langerhans cells, resident immature dendritic cells located within the epidermis and the epithelia of various mucosa, have been shown to be infected following nontraumatic intravaginal exposure to simian immunodeficiency virus (SIV) . Zhang et al.  also demonstrated that activated and resting CD4+ T cells were predominantly infected. Similarly, human studies using mucosal ex-vivo explants have implicated submucosal CD4+ T cells , Langerhans cells [6,9,14] or both cell types . Although those studies demonstrated the presence of provirus in immune cells, they did not describe how HIV is transmitted across the vaginal mucosa then reached the submucosal immune cells in nontraumatic conditions.
Definitive studies of the early infection steps in the human vaginal epithelium are lacking, leading us to the investigation reported here. Indeed, it is still unknown whether HIV is able to cross a pluristratified mucosa as a cell-free viral particle as demonstrated in monostratified mucosa. In this context, by exposing a reconstructed vaginal mucosa to HIV-R5-infected and HIV-X4-infected cells, we established that no translocation of free released virus was detected within the first 4 h following apical inoculation. Furthermore, we demonstrated that Langerhans cells are not capable of enhancing HIV passage during these initial events.
Primary cell cultures
From the Lyon Clinical Investigation Center, according to institutional guidelines, normal human fibroblasts and vaginal epithelial cells were obtained from foreskins and hysterectomy vaginal biopsies, respectively. Primary fibroblast cultures were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL Life Technologies, Grand Island, New York, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco BRL), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis, Missouri, USA). Collagenase (1 mg/ml, 2 h, 37°C, Sigma) and trypsin/ethylenediaminetetraacetic acid (EDTA) (overnight, 4°C) enzymatic treatments were used to isolate vaginal epithelial cells; the cell suspension was cultured in serum-free culture medium [keratinocyte serum-free medium (KSFM), Gibco BRL] supplemented with 50 μg/ml bovine pituitary extract (BPE, Gibco BRL) and 5 ng/ml epidermal growth factor (EGF, Gibco BRL).
Preparation of vaginal mucosa reconstructions
Lamina propria equivalent
Fibroblasts, 3 × 105, were seeded underneath a 12 μm porosity polycarbonate membrane Transwell (Corning Costar Corporation, Portsmouth, New Hampshire, USA), then cultured for 3 weeks in complete DMEM supplemented with 10 ng/ml EGF and 50 μg/ml vitamin C (Aguettant, Lyon, France).
Vaginal mucosa reconstruction
Vaginal epithelial cells 5 × 105, were seeded over the lamina propria equivalent and then cultured for 10 days in modified DMEM/HamF12 (2: 1) medium supplemented with 10% FCS (Hyclone, Logan, Utah, USA). Then, serum concentration decreased from 10 to 1% and CaCl2 (Sigma) concentration raised to 1.7 mmol/l for the following 8 days. During the last 2 days, reconstructions were elevated at the air–liquid interface to improve stratification and differentiation.
Langerhans cells' generation and isolation
CD34+ hematopoietic progenitors, purified from cord blood by magnetic isolation (Miltenyi Biotech, Bergisch Gladbach, Germany), were cultured during 6 days in 200 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (Schering-Plough Research Institute, Kenilworth, New Jersey, USA), 25 ng/ml stem cell factor (SCF) (R&D Systems, Boston, Massachusetts, USA), 2.5 ng/ml tumor necrosis factor-alpha (TNF-α) (R&D Systems) and 0.5 ng/ml transforming growth factor-beta (TGF-β) (R&D Systems). Langerhans cell, 2 × 105, precursors were seeded on the vaginal reconstructions. For confocal microscopy analysis, Langerhans cells precursors were labeled with Orange Cell Tracker CMTMR (Molecular Probes, Leiden, The Netherlands) before seeding. Fresh Langerhans cells isolated from human skin were obtained and characterized as previously described .
Immunohistochemistry analysis and flow cytometry
Normal human vagina and vaginal reconstructions were embedded in Histowax paraffin according to the manufacter's procedure (Intertiles, Brussels, Belgium). Indirect immunohistochemistry stainings were performed on 6 μm sections with a streptavidin–biotin–peroxidase assay (LSAB2 Kit, Dako, Glostrup, Denmark). Negative controls were conducted using unrelated isotype-matched mouse immunoglobulins. Counterstaining was carried out with hematoxylin. The following antibodies were used: anticytokeratin 13 (CBL176, Cymbus Biotechnologies, Chandlers Ford, Hants, UK), anti-Involucrin (NCL-INV, Novocastra, Newcastle upon Tyne, UK), anti-Collagen IV (COL-94, Sigma), anti-Claudin 1 (2H10D10, Zymed, San Francisco, California, USA), anti-Claudin 4 (3E2C1, Zymed), anti-ZO-1 (ZO1-1A12, Zymed), anti-Occludin (OC-3F10, Zymed), anti-CD45 (2D1, BD Biosciences, San Jose, California, USA) and anti-Loricrin (rabbit polyclonal; Babco, Richmond, California, USA). Same antibodies were used for intracellular stainings by flow cytometry analysis as previously described .
Ultrastructural observation by transmission electron microscopy
Vaginal mucosa reconstructions were fixed in 2% glutaraldehyde in cacodylate buffer at 4°C for at least 3 days then postfixed for 1 h with 1% osmium tetroxide in cacodylate buffer with sucrose. After dehydration, they were embedded in epoxy medium. Ultrathin sections were analyzed on a JEOL 1200 EX electron microscope at 80 kV accelerating voltage (Centre des Microstructures, Villeurbanne, France).
Transepithelial electrical resistance and fluorescein isothiocyanate-BSA transmission
Transepithelial electrical resistance (TEER) was measured with a potentiometer accordingly to the manufacter's guidelines (MilliCell; Millipore, Molsheim, France). Fluorescein isothiocyanate (FITC)-BSA passage was evaluated as followed: 100 μl of FITC-BSA (2 mg/ml; Molecular Probes) were added to the apical chamber of vaginal reconstructions. After 4 h of incubation at 37°C, FITC-BSA was removed, basal media were harvested, and then analyzed on a spectrofluorimeter (SpectraMax M5; Molecular Device, Sunnyvale, California, USA).
Reverse transcriptase PCR
Total RNA was extracted from freshly isolated epidermal Langerhans cells, human vaginal epithelial cells and foreskin keratinocytes. Total RNA was extracted using Absolutely RNA Microprep Kit (Stratagene, La Jolla, California, USA) then mRNA was reverse-transcribed to cDNA using Reverse Transcription System (Promega, Madison, Wisconsin, USA). For PCR amplification with Taq DNA polymerase (Promega), primers used are detailed in Table 1. The cycling parameters were as follows: 40 s 94°C/1 min 94°C, 1 min 55°C, 2 min 72°C for a total of 35 cycles.
Virus and peripheral blood mononuclear cell infection
We used the primary isolates 92BR025 (Non Syncitium Inducing, R5) and 91US054 (Syncitium Inducing, X4) obtained from the NIH AIDS Research and Reagent Reference Program. Peripheral blood mononuclear cell (PBMC) were stimulated with phytohemagglutinin (PHA) (1 μg/ml; Sigma) and interleukin (IL)-2 (0.5 ng/ml; Roche, Roche Diagnostics Corporation, Indianapolis, Indiana, USA), and PBMC infection was carried out as previously described . HIV-infected PBMC were harvested at the optimal virus released day. The amount of virus produced by PBMC (revealed by HIV p24 Ag concentration) in 4 h was 2.6 ng/ml for 92BR025 and 9 ng/ml for 91US054 and percentage of infected PBMC were 2–2.5% (p24 positive cells) for 92BR025 and 1.75–1.93% for 91US054.
HIV transmission across vaginal mucosa reconstructions
Reconstructions were exposed to 1 × 106 HIV-infected PBMC. After 4 h of incubation, the inoculum was removed, then basal media were harvested and centrifuged. Supernatants and cell pellets were used either for direct quantification of HIV p24 Ag by ELISA (Innotest HIV Antigen mAb, Innogenetics N.V., Gent, Belgium; detection limit 10 pg/ml) or added to PHA/IL-2-stimulated indicator PBMC for a secondary coculture. Those cocultures supernatants were harvested on days 4, 5 and 7, and analyzed for HIV p24 Ag.
Vaginal mucosa reconstructions containing CMTMR-labeled Langerhans cells alone or inoculated with HIV-infected PBMC labeled with Green Cell Tracker CMFDA (Molecular Probes) were fixed for 40 min with 4% formaldehyde after 4 h of infection. Samples were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes), mounted using Vectashield (Vector Laboratories, Orton Southgate, Peterborough, UK), then analyzed using a Leica TCS SP2 (Centre Commun de Quantimétrie, Lyon, France).
Vaginal mucosa reconstructions and normal human vagina present similar architecture
To evaluate the initial events in HIV entry through the human female genital tract, we developed an in-vitro three-dimensional model of vaginal mucosa using primary human vaginal epithelial cells and fibroblasts (Fig. 1a). Mucosa was reconstructed on a 12 μm porosity Transwell semipermeable membrane to allow cells to migrate freely within the reconstruction. The histological organization of the reconstructed mucosa was first evaluated by immunohistochemistry (Fig. 1b). Fibroblasts seeded underneath the membrane expanded, migrated and synthesized extracellular matrix on both sides of the membrane. Among neosynthesized collagens, Collagen IV was largely found in all the extracellular matrix and not strictly located to the epithelial–chorion membrane zone as in skin's reconstructions . Although reconstructed epithelium was apparently thinner than normal human vagina, specific differentiation markers like cytokeratin 13 and involucrin were clearly expressed. Moreover, as expected, loricrin, a late epidermal differentiation marker found in the skin, was absent (Fig. 1b).
To analyze the role of Langerhans cells in HIV transmission, we integrated dendritic cell precursors (obtained from CD34+ hematopoietic progenitors) in mucosa. As previously described in reconstructed skin, terminal differentiation of CD34+ precursors occurs in the reconstructed tissue . Thus, Langerhans cells could be detected within the recontructed epithelium as demonstrated by the presence of CD45+ cells (Fig. 1c). We, however, noted that Langerhans cell density was lower than in normal human vagina, as previously observed by Regnier et al.  who identified fivefold fewer Langerhans cells in their skin reconstructions than in normal human epidermis. Moreover, to further observe Langerhans cells in vaginal reconstructions, we used Langerhans cell precursors labeled with Orange Cell Tracker CMTMR before seeding. As demonstrated in Fig. 1d, CMTMR-positive Langerhans cells were integrated within the reconstructed vaginal epithelium.
Tight junction proteins are expressed by both vaginal epithelial cells and Langerhans cells
Cell–cell adhesion within stratified epithelium is mediated by adherens junctions and desmosomes. As expected, reconstructed vagina, similarly to normal human vagina, expressed E-cadherin, a typical protein of the cell junctional complex (Fig. 2a). However, tight junctions have been previously described as a hallmark of single-layered epithelia where they seal cells together. Although tight junctions were described in some pluristratified mucosa such as nasal mucosa  or in skin , they have not been detected in human vagina. Tight junction complexes are composed of transmembrane proteins, the Claudin family and Occludin, and of cytoplasmic proteins such as Zonula Occludens proteins (ZO) . In normal human vagina, immunohistochemistry revealed a strong expression of Claudin 1 and Claudin 4 and a low expression of Occludin and ZO-1, as observed in the vaginal mucosa reconstructions (Fig. 2a). However, Claudin 1, Occludin and ZO-1 were not gradiently expressed in reconstructed mucosa due to the thinner epithelium obtained. The presence of tight junction complexes was confirmed by transmission electron microscopy. Indeed, a typical tight junction was observed between two neighboring cells, numerous desmosomes being also present surrounding most epithelial cells (Fig. 2b). Furthermore, we also assessed the TEER (Fig. 2c), an obvious correlation to the presence of functional proteins in the tissue. TEER of mucosal reconstructions reaching 250 Ω/cm2 was lower than TEER of epithelial cell monolayers . Due to the presence of the cornified layer, TEER of skin reconstructions used as positive controls were higher (530 Ω/cm2). On the contrary, TEER of lamina propria equivalent alone were low (123.5 Ω/cm2) almost identical to control TEER (data not shown). Furthermore, we noticed a significant difference between TEER of mucosal reconstructions and mucosal reconstructions containing Langerhans cells. To further rule out the impermeability to macromolecules and to confirm the integrity of the vaginal mucosa reconstructions, we monitored FITC-BSA passage (mean diameter 8 nm) (Table 2). After 4 h, a slight amount of FITC-BSA was measured in vaginal mucosa reconstructions integrating or not Langerhans cells. Furthermore, the presence of Langerhans cells in reconstructed mucosa decreased the amount of FITC-BSA measured after the same time lapse. Therefore, we asked whether Langerhans cells could express tight junction proteins thereby interacting with mucosal epithelial cells. For the first time, we demonstrated that freshly isolated human skin Langerhans cells expressed mRNA of Claudin 1 and ZO-1 (Fig. 3a). mRNA encoding for tight junction proteins by freshly isolated human vaginal epithelial cells and keratinocytes was also illustrated (mRNA of Claudin 1, Claudin 4, Occludin and ZO-1). The expression of tight junction was further detected at protein level by flow cytometry (Fig. 3b). Indirect immunofluorescence analysis revealed that freshly isolated Langerhans cells expressed ZO-1 (Fig. 3c-1). Moreover ZO-1 seemed to be implicated in adhesion point with surrounding keratinocytes, as observed by a bright ZO-1 expression at the contact zone between a large keratinocyte, identified by phase contrast, and a small Langerhans cell (Fig. 3c-2).
Neither R5 nor X4 viral particles cross human vaginal mucosa in 4 h irrespective to the presence of Langerhans cells
Transcellular transport of virus across a pluristratified mucosa remains an open question and its efficiency could be different for either free virions or released virions by infected cells upon contact with the epithelium. No translocation of cell-free HIV inoculated apically was detectable (data not shown) in agreement with Van Herrewege et al.  and as already shown for monostratified epithelia . We therefore inoculated reconstructions with HIV-infected PBMC (Fig. 4a). Briefly, reconstructions were exposed to 1 × 106 92BR025 (R5 virus) or 91US054 (X4 virus) infected PBMC. After 4 h, basal media were collected and centrifuged to separate basal cell-free fractions (supernatants) from basal cell fractions. We first investigated whether translocation of free HIV particles, released upon contact of infected cells with the mucosa had occurred. Hence, basal supernatants were analyzed for both p24 level and subsequent coculture with indicator PBMC up to 7 days. Interestingly, we could not detect significant p24 level for R5 and X4 virus in basal supernatants after 4 h (Table 3), indicating that no transcytosis or passive passage of HIV occurred. In this context, using lamina propria equivalent alone, significant level of p24 were found (1.9 ng/ml) revealing that the mucosal barrier function is not achieved by this subepithelial compartment (data not shown). Observation of infected mucosa by transmission electron microscopy confirmed the absence of free HIV particles within the epithelium, either within epithelial cells or in intracellular spaces (Table 3). In addition, no productive infection could be detected after coculture with indicator PBMC (data not shown). Thus, neither free infectious R5 nor X4 viral particles released by infected PBMC translocated through the vaginal mucosa by paracellular (intercellular) passage or transcellular transport during early infection. Interestingly, no change was observed in the presence of Langerhans cells indicating that they may have no role in the transmission of HIV-free particles across this reconstructed mucosa during the first 4 h of infection.
Langerhans cells may not enhance R5-HIV or X4-HIV transmission and infection during the initial phase of transmission
To analyze more precisely the role of Langerhans cells in the penetration of HIV in this mucosa, we used the same protocol as described above, but instead of analyzing basal supernatants we focused on basal cell fractions. No significant levels of p24 were detected in the basal cell fractions (data not shown). After coculture with indicator PBMC, only a low infection was observed after 5 days of coculture with both virus type (Table 4). Surprizingly, no differences were found in the culture from mucosa containing Langerhans cells or not, indicating that Langerhans cells might not influence HIV transmission in early events. By flow cytometry analysis, and within the limit of sensitivity, no infected PBMC or Langerhans cells were detected in basal compartments after 4 h (data not shown), suggesting that no passive passage by damage within the reconstructed mucosa occurred during the first 4 h of contact with infected PBMC. Furthermore, using a mucosal reconstruction integrating CMTMR-labeled Langerhans cells (red) and inoculated by CMFDA-labeled infected PBMC (green), close interaction between a cell of PBMC inoculum and a Langerhans cell forming a conjugate is observed within the reconstructed epithelium (Fig. 4c). Therefore, our results established that very few infectious virions carried by cells are transmitted in the first 4 h.
To study HIV transmission at the female genital tract, we established a new 3-D human vaginal mucosa model. Our reconstructed mucosa differs from the best-known models. Indeed, in contrast to previous studies using epithelial cell lines  or mimicking only the epithelium compartment [29–31], we developed a model comprising, as in vivo, both the epithelium and the lamina propria using human primary cells. Histological and biochemical data clearly assessed the quality of this mucosa and our model, integrating only Langerhans cells and excluding other immune cell types, allowed studies strictly restricted to the individual role of Langerhans cells in HIV transmission. In this context, previous models have not yet allowed specific analysis of Langerhans cell functions due to the presence of intraepithelial T cells and subepithelial lymphocytes and macrophages [6,9,13,15]. Furthermore, our reconstructed vagina, giving differential and independent access to both apical and basal media, is suitable for the evaluation of HIV and cell migration in contrast to a model developed on dead desepidermized dermis .
In polarized monostratified epithelium, tight junctions form a barrier, which regulates epithelial permeability and paracellular diffusion. For the first time, we demonstrated that human vaginal pluristratified epithelium expressed tight junction proteins, in agreement with previous studies in animals' vagina  or in other human pluristratified tissues [22,23]. We have also provided evidence that human Langerhans cells expressed tight junction proteins, in particular Claudin 1 and ZO-1. Expression of tight junction proteins in dendritic cells, especially in human dendritic cells is poorly described and only Takano et al.  have demonstrated that HLA-DR+ CD11c+ dendritic cells in human nasal mucosa of allergic rhinitis express tight junction proteins. Studies in mouse models established that submucosal intestine dendritic cells express Occludin, Claudin 1 and ZO-1 , skin Langerhans cells express Claudin 1  and a particular mouse subset of lung dendritic cells Langerin+ expresses Claudin 1, Claudin 7 and ZO-2 . To date, tight junction functions in pluristratified epithelium especially in vagina and in dendritic cells are still obscure. An interesting observation is that TEER was higher in the presence of Langerhans cells in the reconstructed mucosa. Similarly, translocation of FITC-BSA in a 4-h period was slower when Langerhans cells are present in mucosa confirming that Langerhans cells may influence the mucosal reconstruction barrier function. Thus, at steady state, Langerhans cells may directly enhance the cohesive structure within the epithelium or may release factors that influence this cohesion.
Previous studies have established efficient transcytosis of HIV across monostratified epithelium. Transcytosis is a rapid transcellular transport of virions. Virions, budding at the contact site of HIV-infected cells and epithelial cells forming a synapse  are internalized into epithelial endosome-like structures then released at the opposite pole of the cells. While transcytosis has been demonstrated in intestinal cell lines , no evidence of this mechanism has been observed in pluristratified mucosa [9,10]. Bobardt et al.  recently demonstrated that a very limited translocation of free virus particles occurred through a pseudomonostratified genital mucosa after 16 h. Therefore, we provided new results showing that no transcellular or paracellular passage of HIV occurs in pluristratified genital mucosa in the first hours of HIV infection.
Numerous studies have attributed to Langerhans cells a crucial role in HIV spreading from mucosal sites in accordance to their location, phenotype and dendritic cells functional properties [6,14,30]. Recent data, however, suggested in complete discrepancy with all previous studies that Langerhans cells do not mediate HIV transmission but rather prevent it . They showed that virions were captured by Langerin and internalized into Birbeck granules then degraded. Therefore, the role of Langerhans cells in HIV transmission across genital mucosa is still ambiguous and further studies should be done to clarify this question. Here, we showed that Langerhans cells alone may not influence HIV transmission during the early infection steps. Nevertheless, the role of Langerhans cells in HIV transmission may be dependent on their density within the vaginal epithelium. Thus, whether Langerhans cell density modulates vaginal HIV transmission, as proposed for foreskin transmission , has to be addressed. Several hypotheses can be proposed to explain HIV transmission across the vaginal mucosa. First, disruption of the vaginal integrity during sexual intercourse or caused by ulcerative lesions may enhance HIV infection . Second, infection may occur by transmigration of infected mononuclear cells present in semen. This mechanism was first decribed by Tan and Phillips  who reported that HIV-infected T cells or monocytes adhere to monolayers of cervical epithelial cells and then transmigrate. Further in-vivo studies confirmed that a limited transmigration of infected mononuclear cells exists through the mice vagina [40,41]. Finally, Langerhans cells or other vaginal immune cells may be involved in HIV transmission. In our experiment, as few Langerhans cells integrated, we were not able to find migrating Langerhans cells in the basal chamber after 4 or 24 h of coculture (data not shown). Nevertheless, although we did not observe CMFDA-labeled infected PBMC in the basal chamber after 4 h, a fraction of infected PBMC was detected after 24 h (6.5% for X4 and 8.3% for R5 virus). Therefore, we propose that after 4 h of coculture, infected PBMC not detectable by flow cytometry may contain cell-associated infectious virus. Thus, we are currently addressing the question of the transmission of HIV at later time point and the precise mechanisms. Finally, a recent paper provided evidence that HIV enters simultaneously Langerhans cells and intra-epithelial T cells. Associated in conjugates, those cells migrate out of the mucosa to disseminate HIV. These interesting results indicate that Langerhans cells and other immune cells such as intra-epithelial T cells are both responsible for HIV spreading . Therefore, the role of Langerhans cells in HIV transmission at later infection stages and the role of infected cells, capable of forming conjugates with Langerhans cells within the vaginal epithelium, remain to be studied.
HIV entry through vaginal mucosa after sexual intercourse represents the initial step of the majority of HIV worldwide infection. In this context, microbicides are urgently needed to prevent this sexual transmission. Unfortunately, different clinical trials testing nonoxynol-9  or cellulose sulfate were suddently interrupted as an increase in HIV infection occurred in the control groups. Thus, to avoid such disappointing experiences, promising microbicides should be preclinically tested on appropriate models to better evaluate any cell cytoxicity. Thus, we present here an interesting and effective model for the evaluation of potential microbicides before their use in clinical trials. Indeed, providing histological and electron microscopy examination, we can evaluate physical disruptions and clues of inflammation, by examining the release of inflammatory cytokines and chemokines in supernatants and analysing Langerhans cell phenotype and functions. In conclusion, our reconstructed vagina will open new perspectives in microbicide exploration and in studying the early HIV infection events.
This work was supported by grants by the Agence Nationale de la Recherche contre le SIDA (ANRS) and SIDACTION. M.B. is supported by ANRS. We also thank ANRS AC31 for helpful discussion.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
HIV infection; Langerhans cells; vaginal mucosa