Thirty-three million people are living with HIV, half of them adult men . Differences in the efficiency of male-to-female HIV transmission versus female-to-male are controversial [2–8] and may depend on several factors thought to influence the risk of female-to-male transmission [6,7,9], including circumcision [7,10–12]. Recent prophylactic trials demonstrate that circumcision could provide more than 50% protection against HIV infection [13–15], but provides no protection to female partners of HIV-positive men [16,17]. Explaining the association between circumcision and reduced HIV acquisition may provide important insight into the mechanism of transmission and development of intervention strategies. Previous reports suggest that a higher density and a more superficial presence of Langerhans cells together with reduced keratinization of the inner layer of foreskin, might increase HIV acquisition in uncircumcised men [18–21]. However, determinants of HIV infection in men are not fully understood and their characterization may aid microbicide development. Current microbicides have been designed to prevent female acquisition [22,23], assuming bi-directional protection ; however, no studies have tested the efficacy of microbicides against penile infection and their potential role in protecting men from insertive vaginal or rectal intercourse.
Using male genital tissue as an ex-vivo model of HIV-1 transmission, we evaluate the frequency of HIV target cells in foreskin, glans and urethra and their differential susceptibility to infection. Furthermore, we evaluate candidate microbicides for safety and efficacy against HIV transmission.
Patients and methods
Patients and tissue
Penile tissue was obtained following gender reassignment at Charing Cross Hospital, London, UK. All participants had ceased hormonal therapy a minimum of 6 weeks prior to surgery. Foreskin tissue was obtained following elective circumcision at St George's University of London, UK. All tissue was collected with written consent according to Local Research committee (LRC) guidelines. Penis and foreskin were cut into 2–3 mm3 explants comprising both epithelium and stroma. Tissue explants were cultured in RPMI 1640 medium supplemented with glutamax, 10% fetal calf serum (FCS), penicillin and streptomycin.
Measurement of tissue viability
Tissue explants were cultured for 10 days, with half medium replacement every 2–3 days. Tissue viability was measured by dimethyl thiazolyl diphenyl tetrazolium salt (MTT) dye reduction as previously described .
Tissue blocks were embedded in optimal cutting temperature compound (OCT), sectioned (12 μm) and stained. Langerhans cells were identified by CD1a ortho kung t6 [(OKT 6) hybridoma (American Type Culture Collection (ATCC)], CD4+ cells by anti-CD4 (clone Q4120, catalogue C1805; Sigma, St Louis, Missouri, USA). Donkey antimouse secondary antibodies were conjugated to Cy3, Cy5, Rhodamine Red-X conjugated (catalogue 715-295-150; Jackson Immunolabs, West Grove, Pennsylvania, USA) and Oregon Green (catalogue 06380; Molecular Probes, Eugene, Oregon, USA). Zenon Alexa Fluor 647 (catalogue Z25008; Molecular Probes) was utilized for antibody illumination. Epithelium was stained with wheat germ agglutinin (WGA) conjugated with Alexa fluor 594 (catalogue W11262; Molecular Probes).
Imaging of tissue slices
Images were collected on a DeltaVision RT system using a 40× oil objective, an Olympus IX71 microscope, and analysed using deconvolution microscopy software. Thirty z-sections, 0.5 μm apart, were collected per image field. All measurements were obtained through the ‘Measure Distances’ tool, using a standard two-point method.
Determination of microbicides cytotoxicity
Compounds used in this study were Cyanovirin-N (Biosyn Inc.; Huntingdon Valley, Pennsylvania, USA), PRO 2000 (Indevus Pharmaceutical, Lexington, Massachusetts, USA) and poly(methyl 2-propionamidoacrylate) (PMPA) (Gilead Sciences, Foster City, California, USA). Cyanovirin-N was provided unformulated, whereas PRO 2000 and PMPA were gel-formulated compounds. Placebo formulations containing the same excipients as the active product were also provided. All compounds were diluted into culture media. Potential toxicity of microbicides was measured as previously described .
Cells and viral cultures
PM1 cells (AIDS reagent project, NIBSC, Potters Bar, Hertfordshire, UK) were cultured in RPMI 1640 medium supplemented with glutamax, 10% FCS, penicillin and streptomycin. HIV-1BaL and HIV-1LaV were grown on peripheral blood mononuclear cells (PBMCs) as previously described  and the TCID50 determined.
HIV infection of human male genital tract tissue explants and dissemination by migratory cells
Tissue explants were exposed to HIVBaL or HIVLaV (104 TCID50/explant) for 2 h. Aldithriol-2 (10 mmol/l)-treated virus and medium only were used as negative controls. After washing with phosphate-buffered saline (PBS), explants were resuspended in medium ± phytohaemagglutinin (PHA; 10 μg/ml) overnight. The day after, tissue explants were removed and cultured ± PHA for 3 days, replaced by IL-2 (100 IU/ml) for the following 8 days. Migratory cells present in overnight culture plates were washed with PBS and cocultured with PM1 (40.000 cells/well) for 7 days. Culture supernatants were harvested every 2–3 days and assessed for p24 antigen level (HIV-1 p24 Antigen EIA, catalog # 626391, Beckman Coulter, High Wycombe, UK). Statistical analysis was performed using two-tailed t-test.
Screening of candidate microbicides
Tissue explants were treated with medium or compound just prior to exposure to HIV-1BaL (104 TCID50) for 2 h in presence of the compound. The samples were then washed with PBS and cultured in the absence of compound. The day after, migratory cells were separated from tissue as described above. Cultures were assessed for viral replication by p24 enzyme-linked immunosorbent assay (ELISA) as described.
Tissue samples were cultured with or without compound for 2 h, washed with PBS and cultured in medium overnight. Supernatant was then collected and 23 cytokines quantified by in house multiplex bead immunoassay as described .
Establishment of optimal culture conditions
Human male genital explant cultures were established by adapting previous methods developed for culture of cervicovaginal tissue . Viability of explants in culture was assessed (data not shown). Glans, meatus and urethra maintained high viability for up to 7 days (average viability: 76.5–97.8%), with a slight decline by day 10 (62.7–76.7%). Inner and outer foreskin demonstrated shorter viability (75% at day 3 but 38% by day 7).
HIV target cell distribution
Genitourinary tissue was sectioned and stained with fluorescent antibodies to identify Langerhans cells and CD4+ cells following 0, 3 or 7 days in culture (Fig. 1a, b). Fluorescent WGA was used to reveal tissue structure and integrity.
For each target cell identified, we measured epithelial thickness, distance of the cell body to tissue surface and distance of the closest Langerhans cell projection to tissue surface (Fig. 1c). Epithelial thickness was determined by measuring the distance between the basement membrane and tissue surface, crossing through each cell body. Following 7 days in culture, specimens of inner foreskin were severely fragmented and lacked semblance of integrity. We, therefore, focused on differences in tissue from the initiation to 3 days in culture.
Overall, average epithelial thickness for each tissue type remained similar throughout the 3-day culture period (Fig. 2a), although inner foreskin decreased (100.4–92.4 μm) and glans epithelium increased in thickness (95.8–106.9 μm) after 3 days.
Similarly, the average distance of Langerhans cell bodies to tissue surface decreased in both inner (73.4–66.2 μm) and outer foreskin (71.6–61.3 μm), and increased in glans (57.3–66.7 μm) epithelium after 3 days in culture (Fig. 2b). Upon examination, we found no change in Langerhans cell projection distance to tissue surface over time for inner foreskin (Fig. 2c). However, Langerhans cell projections appeared to move closer to the tissue surface in outer foreskin (59.5–47.7 μm) and farther in glans (41.4–54.8 μm). These changes most likely reflect an increase in epithelial thickness observed with time.
Next we enumerated the number of Langerhans cells observed in each image. Although no difference in Langerhans cell number was observed in inner or outer foreskin samples after 3 days (Fig. 2d), for glans tissue we observed that the value decreased from 0.033 to 0.017 Langerhans cells/100 μm2 after 3 days. In addition, a significantly higher number of Langerhans cells were observed in glans compared with inner and outer foreskin, and in inner compared with outer foreskin, with an average of 0.023 and 0.017 Langerhans cells/100 μm2 in inner and outer foreskin, respectively. These values decreased to 0.02 and 0.016 Langerhans cells/100 μm2 by day 3.
The majority of CD4+ cells observed in the tissue specimens typically reside below the basement membrane in mucosal tissue, but may infiltrate surface epithelium under inflammatory conditions , whereas CD4+ Langerhans cells exclusively reside in the epithelium. No change was observed for CD4+ cell number for inner foreskin over 3 days in culture, but we detected a slight increase in outer foreskin, from 0.01 to 0.016 cells/100 μm2 (Fig. 2e). In contrast, significantly fewer CD4+ cells were found in glans following 3 days in culture. Here, we observed 0.037 CD4+ cells/100 μm2 imaged in glans tissue at the initiation of culture, decreasing to 0.013 cells/100 μm2 by day 3.
When the density of CD4+ cells was compared between tissue sites, glans tissue was found to contain the most CD4+ cells/100 μm2 at the initiation of the culture (Fig. 2e), whereas inner foreskin had 0.026 cells/100 μm2 or 2.6 times more than outer foreskin. By 3 days in culture, the only difference still detected was between inner foreskin and glans, with inner foreskin containing 1.6 times more CD4+ cells/100 μm2.
This trend continued for CD4+ cells observed within surface epithelium (Fig. 2f). Whereas no significant differences were observed in the number of CD4+ cells/100 μm2 of surface epithelium for either inner or outer foreskin, glans tissue exhibited a 2.7-fold decrease, from 0.0097 to 0.0037 after 3 days. In addition, glans showed significantly greater numbers of CD4+ cells in the epithelium compared with both inner (0.0019 CD4+ cells/100 μm2) and outer foreskin (0.0009 CD4+ cells/100 μm2) at the initiation of culture.
Despite these differences in cell number, the average distance of epithelial CD4+ cells to tissue surface remained unchanged in all tissue types throughout the culture period (Fig. 2g).
HIV infection of male genital tissue
Ex-vivo susceptibility to HIV-1 infection and relative efficiency of HIV dissemination by migratory cells were determined by exposing glans, urethra and foreskin to either HIV-1BaL (R5 isolate) or HIV-1LAV (X4 isolate). HIV-1BaL productively infected all tissue sites investigated (Fig. 3a), but no infection was detected with HIV-1LAV. Cellular emigrants from all tissue sites (±PHA) were able to disseminate HIV (BaL and LAV), but the level of HIV-1LAV p24 in culture supernatant was generally lower compared with HIV-1BaL (Fig. 3b,c). No significant differences in the level of HIV-1 infection either between different tissue sites or between activated versus nonactivated tissue and migratory cells were detected.
Cytokine release profile of male genital tissue explants
As cytokines can significantly modulate tissue susceptibility to HIV infection, the pattern of cytokine release from glans and foreskin after 24 h in culture was analysed (Fig. 4). IL-1-α, IL-4, IL-12, IL-15, tumour necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) were all below or at the limit of detection for all three tissue sites. Among the cytokines produced at moderate levels, interferon-β (IFN-β), IFN-γ, monokine induced by γ-interferon (MIG), stromal cell derived factor 1-β (SDF-1-β), granulocyte-macrophage colony-stimulating factor (GM-CSF) and monocyte chemotactic protein (MCP-2) levels were significantly higher for both inner and outer foreskin compared with glans (P < 0.05), whereas IL-2 levels were only higher for outer foreskin compared with glans (P = 0.035). The same was observed for IL-6, IL-8, macrophage inflammatory protein (MIP-1-β), MCP-1 and interferon-γ-induced protein (IP-10) (P < 0.05) released at higher concentrations, whereas MIP-1-α and G-CSF were only released at significantly higher levels by inner foreskin (P < 0.01) when compared with glans. Inner and outer foreskin had a comparable pattern of cytokine release, with the exception of G-CSF, in which secretion by inner foreskin was significantly higher than outer foreskin (P = 0.006). Overall, both inner and outer foreskin demonstrated a significantly higher level of production of most secreted cytokines when compared with glans.
Evaluation of microbicide safety and efficacy
PRO 2000, PMPA and Cyanovirin-N were tested for their ability to inhibit HIV-1BaL infection and dissemination (Fig. 5). PMPA and PRO 2000 gel placebo demonstrated no activity against HIV infection (data not shown). No toxicity was detected at the highest concentrations tested for any of the compounds (data not shown).
PMPA at 1 mg/ml (10-fold dilution of the original 1% stock gel) inhibited HIV infection of glans by 90%, and completely inhibited dissemination by migratory cells (Fig. 5a). PRO 2000 at 100 μg/ml (400-fold dilution of the original 4% stock gel) inhibited infection by 99.4%, and dissemination of infection was totally suppressed (Fig. 5b). Cyanovirin-N at 11 μg/ml prevented HIV replication in tissue (95%) and propagation of the virus by migratory cells (99%; Fig. 5c). Similar results were obtained when tested on foreskin (data not shown).
PMPA, PRO 2000 and Cyanovirin-N had little effect on the 23 cytokines tested and none affected cytokine release by outer foreskin. However, Cyanovirin-N at 11 μg/ml increased production of SDF-1-β (2.8-fold) and MIP-1-β (2.9-fold) in glans, and IL-2 (two-fold), IFN-β (2.4-fold), IFN-γ (1.6-fold), MIG (21.2-fold), GM-CSF (1.4-fold), IP-10 (2.7-fold) and MCP-2 (1.8-fold) in inner foreskin (P < 0.05). PRO 2000 at 100 μg/ml did not induce any modification in cytokine release by both inner and outer foreskin, but reduced the production of SDF-1-β (2.3-fold; P < 0.01), MIP-1-β (six-fold), IP-10 (3.1-fold) and MCP-2 (2.7-fold; P < 0.05) in glans. PMPA 1 mg/ml did not exert any significant variation in cytokine release for all tissue sites tested, with the only exception of inner foreskin, where SDF-1-β release increased two-fold (P < 0.05).
All male genital tissue sites examined adapted well to explant culture, with the exception of foreskin, that lost tissue integrity after 3 days, most likely reflecting that foreskin epithelium is supported by a thinner layer of stroma compared with the other tissue sites. Interestingly, foreskin explants released higher levels of cytokines compared with glans, suggesting a higher level of immune activation. At the initiation of culture, the average number of Langerhans cells and CD4+ cells was greatest for glans > inner foreskin > outer foreskin, in agreement with a previous report  comparing inner and outer foreskin. However, another study reported a higher density of CD4+ cells and Langerhans cells in outer foreskin > inner foreskin > glans . These discrepancies might reflect that in the latter study, tissue was obtained postmortem or that foreskin and glans tissue in this study were not taken from the same donors.
The distance of dendritic projections emanating from Langerhans cell bodies was closest to the epithelial surface for glan > inner = outer foreskin. These observations also differ to those of McCoombe and Short , in which Langerhans cell bodies and projections were particularly superficial in inner foreskin. In agreement with both previous studies [18,19], CD4+ cells in all tissue sites were predominantly distributed within the stroma underlying the epithelium. Significant changes in cell number and dendritic projections were observed in culture. The most pronounced being the decrease in CD4+ and CD1a+ cells in glans by day 3, suggesting a considerable migration of both CD4+ cells and Langerhans cells, out of the tissue. In contrast, the number of Langerhans cells and CD4+ cells in the epithelium of both inner and outer foreskin were fairly stable.
All genital tissue sites examined were susceptible to R5 HIV-1BaL infection, independent of immune activation. In contrast, no infection was observed with X4 HIV-1LAV. These data confirm a previous study of human foreskin  and reflect the predominant CCR5 expression in foreskin and cervical tissue [19,29,30]. However, this is not the case for penile glans and urethral meatus, reported to have equal levels of CCR5 and CXCR4 expression [18,19]. Interestingly, migratory cells from all male tissue sites also preferentially transmitted HIV-1BaL over HIV-1LAV, in agreement with previous observations that in vitro derived Langerhans cells and dendritic cells preferentially transmit R5 virus [31–38].
To investigate the potential role of male genital explants as a model for microbicide development, we selected three microbicide candidates: PRO 2000, which is in phase III efficacy trials [39–41]; PMPA gel (tenofovir), which is in phase II trials (, www.microbicides.org); and Cyanovirin-N, which is in preclinical development [43–46]. PRO 2000 at 100 μg/ml (1/50 of the vaginal gel) provided nearly complete suppression of viral infection and dissemination in penile tissue, with no toxicity and little modulation of cytokine expression. These data are in agreement with previous studies of cervical and rectal tissue [25,26,47] and with a clinical study demonstrating safety for 4% gel applied daily on the penis . PMPA at 1 mg/ml (1/10 of the vaginal gel) demonstrated potent activity against R5 HIV-1BaL infection of glans tissue and dissemination of virus. However, it was less active than a previous study using colorectal explants . PMPA demonstrated no toxicity for penile tissue and little modulation of cytokine expression, in agreement with human studies demonstrating good tolerability of PMPA gel 1% applied vaginally . Cyanovirin-N conferred 95% protection against HIV-1BaL at 11 μg/ml similar to that seen in cervical explants  and at a dose 1/500 of that shown to be protective in macaques [45,46]. The ability to block HIV-1 transfer by migratory cells was in agreement with the ability of Cyanovirin-N to impair dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)-mediated HIV-1 transmission in vitro. Although Cyanovirin-N was not toxic for male genital tissue explants, we observed a degree of cytokine disregulation following 2 h exposure to Cyanovirin-N, in line with previous reports .
In summary, this study describes an ex-vivo model to study the vulnerability of male genital mucosa to HIV infection. Although we observed some differences in the frequency of target cells (CD4+ and Langerhans cells) between tissue sites, all sites examined were susceptible to HIV-1 (R5) infection. We found no evidence to support enhanced susceptibility of inner foreskin relative to glans and outer foreskin. Nevertheless, circumcision would remove two out of three of the exposed surface areas of the penis, reducing the chance of virus coming in contact with susceptible target cells. The susceptibility to infection and the observed inhibition by three different microbicides, two of which are already in clinical trials, offer new perspectives for the development of compounds able to protect male genital tissue from HIV transmission.
We are most grateful to Mr James Bellringer, Consultant Urologist and Gender Surgeon of Charing Cross Hospital, London, for provision of penile tissue samples and to St George's Hospital, London for providing the foreskin tissue, as well as the many tissue donors who have contributed to this work. We also would like to thank Ms Naomi Louise Armanasco for coordinating the tissue specimen collection.
R.J.S. and L.F. designed the experiments; L.F. performed the cellular and explant experiments; S.M.B. and T.J.H. performed all the immunohistochemistry and imaging analysis; R.J.S., L.F. and T.J.H. wrote the article.
The present work was funded by the National Institutes for Health Grant R21 HDO 48391-01. LF is supported by Grant Number F30DA023367 from the National Institute On Drug Abuse. We also acknowledge the generous grant from the Fondation Dormeur that facilitated purchase of equipment used in this project.
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