Background:  Male circumcision has been shown to reduce rates of HIV acquisition in men in clinical trials, but few studies have explored the biological mechanism behind this protective effect, and even these have yielded conflicting results. Utilizing advanced immunofluorescence microscopy, our laboratory has successfully visualized the penetration of infectious HIV-1 particles into explanted tissue. We report here the findings from a study of 15 adult male foreskins that confirm the ability of HIV-1 to penetrate stratified squamous epithelium and infect underlying target cells.

Methods:  Adult male foreskins were obtained from consenting male donors in Chicago, Illinois. The specimens were separated into inner and outer foreskin and dissected into smaller sections. Sections were inoculated with photoactivatable GFP-Vpr HIV_Bal or HIV_R7 and incubated for short-term (4 to 24 hours) or long-term (3 days) studies. Negative controls were included with media alone. After set incubation times, specimens were flash-frozen and thin sections fixed and stained with WGA, DAPI, or antibodies against HECD1, ZO-1, p24, or Langerin. Images were captured and analyzed using DeltaVision RT microscopy systems and SoftWorx software. Images were analyzed for photo-activated GFP-Vpr HIV-1 particles and Langerhans cells (LC). Analysis was conducted with SPSS Software 16.0.

Results:  Equal depths of viral penetration were observed in inner and outer foreskin. Over 80% of penetrating viral particles were located interstitially. The presence of intercellular junctions in deeper strata was constant between the inner and outer foreskin. Viral penetration was seen at depths where superficial LC were located within the epithelium. Dual-labeled infected LC were also identified in the tissue in long-term explant studies.

Conclusions:  We have visualized HIV-1 particles breaching epidermal barriers and penetrating into both inner and outer foreskin. HIV-1 was seen penetrating to depths where high numbers of LC are found, and infected LC were clearly identified in the tissue. This supports the notion that LC can play a direct role in mediating initial infection of HIV-1 in the male genital tract. Future studies characterizing viral interactions with other target cells as well as with penile tissue will continue to contribute to a better understanding of how HIV is sexually transmitted in men.